kir–hla receptor-ligand mismatch associated with a graft-versus-tumor effect in haploidentical...
TRANSCRIPT
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KIR–HLA Receptor-Ligand Mismatch Associated With a Graft-Versus-Tumor Effectin Haploidentical Stem Cell Transplantation for Pediatric Metastatic Solid Tumors
Antonio Perez-Martınez, MD, PhD,1* Wing Leung, MD, PhD,2 Evangelina Munoz,1 Rekha Iyengar, PhD,2
Manuel Ramırez, MD, PhD,1 Jose Luis Vicario, PhD,3 Alvaro Lassaletta, MD,1 Julian Sevilla, MD, PhD,1
Marta Gonzalez-Vicent, MD, PhD,1 Luis Madero, MD, PhD,1 and Miguel Angel Dıaz-Perez, MD, PhD1
INTRODUCTION
Little progress has been made in the survival of children with
metastatic and refractory solid tumors [1]. Allogeneic hemato-
poietic stem cell transplantation (HSCT) has been proposed as a
potential curative alternative in patients with refractory malignan-
cies [2]. A possible graft-versus-tumor (GVT) effect has been
documented in children with metastatic and relapsed Ewing
sarcoma [3], neuroblastoma [4], melanoma [5], and hepatoblastoma
[6]. However, the immunological mechanisms that mediate the
GVT effect have not been elucidated. Early immune reconstitution
after a conditioning regimen is led by natural killer (NK) cells, and
for several weeks, they represent the only detectable lymphoid
population. NK cell alloreactivity in vitro may be predicted by the
lack of ligands for inhibitory killer immunoglobulin-like receptors
(KIRs) [7]. Inhibitory KIRs bind with four specificities for
polymorphic human leukocyte antigen (HLA) class I molecules.
NK cell reactivity may be further enhanced by a Th1 cytokine
environment and by T-cell lymphopenia [8,9].
To evaluate the potential of NK cell-mediated GVT effect and
its immunological mechanisms, we performed a pilot study of
haploidentical HSCT in three children with refractory tumors with
no known cure.
METHODS
Patients and Transplantation
The study involved three patients with metastatic solid
tumors that were refractory to chemotherapy (Table I). This
study was approved by the Ethical Committee of Hospital Nino
Jesus. Informed consent was obtained in accordance with the
Helsinki Declaration. HSCT was performed using parental CD3/
CD19 depleted G-CSF mobilized peripheral blood. Reduced-
intensity conditioning consisted of busulfan (4 mg/kg/day, 2 days),
fludarabine (30 mg/m2/day, 5 days), thiotepa (5 mg/kg/day, 2 days),
and methylprednisolone (4 mg/kg/day, 5 days). Methotrexate
(15 mg/m2/day, day þ1, and 10 mg/m2/day, days þ3 and þ6) and
cyclosporine (3 mg/kg/day, day�1 until 1 month after the infusion)
were also administered. Immune reconstitution and hematopoietic
chimerism were assessed monthly after HSCT. Computed tomo-
graphy andmagnetic resonance imagingwere performed before and
3 months after HSCT.
HLA Typing and KIR Genotyping, Phenotyping,and Cytotoxicity
HLA-C allotypes (C1 andC2) andHLA-B allotypes (Bw4) were
determined using high-resolution polymerase chain reaction-
Killer immunoglobulin-like receptors (KIRs) on natural killer cells(NKs) recognize groups of human leukocyte antigen (HLA) class Ialleles. Cells without an inhibitory HLA ligand may trigger NKactivation. Reduced risk of relapse has been reported in malignanthematologic diseases after haploidentical transplantation when HLAligands against the inhibitory KIRs present in the donor were absentin the recipient. We performed haploidentical transplant in three
children with refractory solid tumors. Our results showed thatbeneficial antitumor effects could be observed in the presence ofinhibitory KIR–HLA mismatch. These preliminary results suggest apossible association between disease control and NK cell allor-eactivity. Pediatr Blood Cancer 2009;53:120–124.� 2009 Wiley-Liss, Inc.
Key words: haploidentical stem cell transplantation; KIR-HLA mismatch; NK cells; pediatric solid tumors
——————1Department of Pediatric Hematology and Oncology, Hospital Nino
Jesus, Universidad Autonoma de Madrid, Madrid, Spain; 2Department
of Oncology, St. Jude Children’s Research Hospital, Memphis,
Tennessee; 3Laboratorio de Histocompatibilidad y Biologıa
Molecular, Centro de Transfusion de Madrid, Madrid, Spain
Grant sponsor: Spanish National Health Service; Grant number: FIS
CM-04/00011.
*Correspondence to: Antonio Perez-Martınez, Servicio de Oncologıa y
Trasplante del Hospital Infantil Universitario Nino Jesus, Menendez
Pelayo 69, Madrid 28009, Spain.
E-mail: [email protected]
Received 7 November 2008; Accepted 6 January 2009
� 2009 Wiley-Liss, Inc.DOI 10.1002/pbc.21955Published online 12 February 2009 in Wiley InterScience(www.interscience.wiley.com)
120 Brief Reports
sequence-based typing. Fifteen human KIR genes and two
pseudogenes were analyzed by polymerase chain reaction with a
KIR typing kit (MiltenyiBiotec, Inc., Auburn,CA).Antibodies used
to identify KIR phenotypes by flow cytometry were CD45-FITC,
CD19/CD20-PE, CD56-APC, and CD158a,h (EB6)-PE (Beckman
Coulter, Fullerton, CA); CD3-PECY7 and CD158b1/b2,j (CHL)-
FITC (Becton Dickinson, Franklin Lakes, NJ); and CD158e1
(DX9)-FITC, CD158e1 (DX9)-PE, and CD158a,h (EB6)-FITC
(Miltenyi Biotec, Inc.). Natural cytotoxicity of donor unstimulated
fresh NK cells against K562 leukemia cells was assessed in a
conventional 2-hr europium-TDA release assay (Perkin-Elmer
Wallac, Turku, Finland). NK cells were magnetically isolated from
peripheral blood of donors using standard density gradient
centrifugation over Ficoll-Paque Plus (Pharmacia, Uppsala,
Sweden) and NK cell isolation kit from Miltenyi Biotec, Inc.,
following the manufacturer’s specifications. Labeled cells were
processed on a QuadroMACS separator with LS columns (Miltenyi
Biotec, Inc.). Purity was higher than 95% of CD56þCD3� cells.
RESULTS
Patient 1
A 4-year-old male with an embryonal rhabdomyosarcoma of
the right testicle and lung metastasis underwent surgery and
chemotherapy according to the Malignant Mesenchymal Tumor
Committee MMT 98 protocol [10]. Because lung metastasis
persisted, he received second-line chemotherapy with topotecan/
cyclophosphamide and then third-line chemotherapy with irinote-
can/temozolomide, resulting in partial response. HSCT was then
performed using a parental donor mismatched at KIR2DL1 with
missing HLA-CLys80 in the recipient. In addition, donor NK cells
showed high lysis to K562 cells in vitro and possessed three
activating genes KIR2DS1, KIR2DS3, and KIR3DS1 (Fig. 1A–C);
the latter twomay interact with the HLA-CAsn80 and Bw4 present in
the recipient. The patient achieved full donor chimerism 1 month
after transplantation, and cyclosporine was withdrawn. Ten days
later, the patient presented with herpes zoster infection and
pancytopenia. Bone marrow chimerism was 98% recipient.
Secondary graft failure was diagnosed and treated with steroids,
cyclosporine, and granulocyte colony-stimulating factor. Because
no responsewas observed, a secondHSCT from the same donor was
performed 3 months later, adding muromonab (an anti-CD3
monoclonal, 0.1 mg/kg/d, 7 days) to the conditioning regimen
described above. Full donor chimerism was reached 1 month after
the second HSCT, and no toxicities were observed. Since then, NK
cells were the predominant lymphocytes throughout the first year
after transplantation and lung metastasis no longer appeared in
computed tomography evaluations (Fig. 1D). Ayear and a half after
Pediatr Blood Cancer DOI 10.1002/pbc
TABLE I. Patients Characteristics and Outcome
Subject Patient 1 Patient 2 Patient 3
Tumor Embryonal rhabdomyosarcoma Neuroblastoma Ewing sarcoma
Metastasis Lung Bone/BM Bone /BM
Chemotherapy MMT IV 98.11 HR-NBL’1/ESIOP SEOP 2001
Surgery Yes Yes Yes
Radiotherapy No Yes No
Second lines chemotherapies TPT-CCF/IRT-TMZ TVD/IRT-TMZ ICE/TPT-CCF/IRT-TMZ
High dose chemotherapy No Yes No
Pre-HSCT status Partial remission Metastatic relapse Metastatic relapse
Graft content: CD34/CD3/CD56 (�106/kg) 5.7/0.2/9 5.2/0.34/60 5.8/0.8/17
Toxicities Zoster None None
NE/PE (day) 12/15 13/14 15/16
Post-HSCT response Complete remission No response Partial response for 5 months
Outcome/time after HSCT Alive/21þ months DOPD/10 months DOPD/9 months
HLA-B
Patient 44/52 (Bw4/4) 49/55 (Bw4/6) 51/41 (Bw4/6)
Donor 44/49 (Bw4/4) 44/49 (Bw4/4) 41/41 (Bw6/6)
HLA-Cw
Patient 12/16 (Asn80/Asn80) 3/6 (Asn80/Lys80) 15/17 (Lys80/Lys80)
Donor 7/16 (Asn80/Asn80) 6/6 (Lys80/Lys80) 7/17 (Asn80/Lys80)
KIR2DL1 (%)
Patient — — —
Donor 12.6 6 10.9
KIR2DL2/3 (%)
Patient — — —
Donor 28.2 25.2 33.8
KIR3DL1 (%)
Patient — — —
Donor 2.5 3.4 22.3
KIR–HLA
Receptor-ligand No Lys80 All ligands present No Asn80
Mismatch KIR2DL1 mismatch No KIR–HLA mismatch KIR2DL2/3 mismatch
BM, bone marrow; NE, neutrophil engraftment; PE, platelet engraftment; TPT-CCF, topotecan cyclophosphamide; IRT-TMZ irinotecan-
temozolomide; TVD, topotecan-vincristine-doxorubicin; ICE, ifosfamide-carboplatin-etoposide; DOPD, died of progressive disease; HSCT,
haploidentical stem cell transplantation.
Brief Reports 121
Pediatr Blood Cancer DOI 10.1002/pbc
Fig. 1. KIR NK cell profile, immune reconstitution and clinical response after haploidentical stem cell transplantation. A: KIR inhibitory donor
phenotype and KIR donor genotyping. B: Patient’s immune reconstitution after haploidentical HSCT. C: Donor’s NK cytotoxicity against K562
cells.D: Lung computed tomography of Patient 1withmetastatic rhabdomyosarcoma at diagnosis and after chemotherapy. The far right panel shows
the absence of metastatic lesions in the lung 1 year after HSCT.E: Short tau inversion recovery saggital magnetic resonance images of lumbar spine
of Patient 3. The left panel, before HSCT, shows several focal bone marrow lesions, most pronounced at L2–L3. The right panel shows complete
resolution of the lesions and fatty replacement of the vertebral bone marrow, indicating a positive response.
122 Brief Reports
the second HSCT, the patient was still alive with full donor
chimerism, donor NK genotype, high lysis to K562 cells and
complete remission.
Patient 2
A 16-year-old female with a stage IV suprarenal neuroblastoma
and metastasis to bone and marrow was treated according to the
Societe Internationale d’Oncologie Pediatrique HR-NBL-1/ESIOP
protocol [11]. Chemotherapy was followed by primary tumor
resection. Evaluation after treatment showed progressive disease in
bone marrow and suprarenal gland. Because first-line treatment
failed, three cycles of topotecan/vincristine/doxorubicin (TVD) and
high-dose chemotherapy (busulfan/melphalan) with autologous
stem cell rescuewere administered. Partial remission was achieved,
but 3 months later, progressive disease was observed in the dorsal
column, bone marrow, and right suprarenal gland. Three cycles of
TVD plus third-line chemotherapy (irinotecan/temozolomide)
were given with minimal response. HSCT was then perform-
ed with a parental donor with no inhibitory KIR–HLA
mismatch. Furthermore, the donor NK cells had very poor lytic
activity against K562 cells in vitro and possessed only two
activating genesKIR2DS2 and 2DS3 but notKIR2DS1orKIR3DS1
(Fig. 1A–C). Full donor chimerism was reached 1 month
after HSCT, and no toxicities were observed. NK cells were the
most numerous during the first 3 months after transplantation
with rapid reconstitution of CD3þ lymphocytes thereafter. Her
tumors had no objective response after HSCT and progressed at
6 months in the dorsal and lumbar regions associated with medullar
compression. The patient died of neuroblastoma 10 months
after HSCT.
Patient 3
A 17-year-old male received chemotherapy according to the
Spanish Society of Pediatric Oncology SEOP-2001 protocol for
group 1 right fibula Ewing sarcoma. The regimen consisted of an
induction phase of vincristine/ifosfamide/etoposide/doxorubicin,
right lower extremity amputation, and a consolidation phase of
vincristine/actinomycin/cyclophosphamide. Seven months after
treatment, relapsed disease was found in bone marrow and bone
(lumbar spine and humerus). Second-line chemotherapy (ifosfa-
mide/carboplatin/etoposide) was given without response. He then
received third-line chemotherapy with two cycles of topotecan/
cyclophosphamide and two cycles of irinotecan/temozolomide.
Despite persistent disease, HSCT was performed using a parental
donor mismatched at KIR2DL2/3 with missing HLA-CAsn80 in the
recipient. Donor NK cells showed intermediate natural cytotoxicity
against K562 cells and possessed three activating genes KIR2DS1,
KIR2DS2, and KIR3DS1 (Fig. 1A,C); the latter two may interact
with the HLA-CLys80 and Bw4 present in the recipient. Full donor
chimerism was reached 1 month after HSCT, and no toxicities
were observed. In the first 3 months after transplantation, NK cells
were the predominant lymphocyte population and a total response in
the vertebral bones were observed (Fig. 1E). CD3þ lymphocytes
then rapidly reconstituted in the following 2 months, with falling
NK cell counts and progressive tumors in lumbar spine, humerus,
and lung appeared 5 months after transplantation. The patient died
of progressive disease 9 months after HSCT.
DISCUSSION
Clinical response has been observed in previous pediatric solid
tumor allogeneic transplantations. Although a GVT effect was
suggested (in addition to the conditioning chemotherapy effect), the
actual immune mechanisms have never been elucidated. For
instance, graft-versus-neuroblastoma effect has been suggested
in haploidentical transplantation for advanced disease [5,12].
Refractory metastatic Ewing sarcoma and rhabdomyosarcoma has
been treated with allogeneic transplant [4,13,14]. An immuno-
logical effect associatedwith graft-versus-host disease (GVHD) has
been described in hepatoblastoma [6], and a rapid recovery of NK
cytotoxic activity was describe in successful cases of metastatic
osteosarcoma after allogeneic bonemarrow transplantation [15,16].
The results of our preliminary study suggest that a clinically
beneficial GVT effect might be mediated by donor–recipient
inhibitory KIR–HLA mismatched NK cells. Clinical response was
observed in the two patients with a KIR–HLA mismatched donor
during the time when NK cells were the major lymphocyte
population. In addition, the degree of tumor response appeared to
correlate with the donor NK natural cytotoxicity [17], the disease
status at the time of transplantation, the number of KIR activating
receptors (2DS1, 2DS3, 3DS1), and possibly with the presence of
activating KIR3DS1–Bw4 interaction. These findings raise the
possibility that KIR is a major biological determinant of antitumor
effect in pediatric cancer, in addition to hematologic malignancies
as demonstrated in adult AML by the Perugia group [18,19] and in
pediatric AML and ALL by the Memphis group [20].
Our observation that tumor responses were only seen in those
patients with inhibitory KIR–HLA mismatched NK cells and
provide suggestive evidence that allogeneic NK cells might be
the effector cells that mediated GVT effects in the setting of a
mismatched allogeneic transplant. Although our results are
preliminary, this report provides novel data to support further
investigation in the use of KIR–HLA mismatched HSCT for the
treatment of refractory childhood solid tumors.
ACKNOWLEDGMENT
The authors want to thank Dr. Sara Sirvent and Dr. Gustavo
Albi for computed tomography andmagnetic resonance images, and
Dr. David Galloway for scientific editing. The authors also want
to thank Fundacion Inocente for supporting the project ‘‘Allogeneic
Stem Cell Transplantation in Pediatric Solid Tumors.’’ A.
Perez-Martınez was supported by Spanish National Health Service
grant FIS CM-04/00011.
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Congenital Glioblastoma
G.M. Milano, MD, PhD,1* C. Cerri, MD,1 V. Ferruzzi,1 I. Capolsini, MD,1 E. Mastrodicasa, MD,1
L. Genitori, MD,2 and F. Aversa, MD1
INTRODUCTION
First described by Holt [1], congenital brain tumors, which
account from 0.3% to 4% of CNS tumors in children are extremely
rare forms of childhood tumors. They often arise during pregnancy
and are identified only at autopsy [2,3]. In the century since the first
report, the entire cohort comprises nearly 600 cases, all of which
had a very poor prognosis [4]. Characteristics and outcomes of
200 congenital brain tumors were extensively discussed in a review
by Wakai et al. [7].
The Solitare and Krigman classification groups congenital
brain tumors according to time of diagnosis as: definitely congenital
(present at birth), probably congenital (detected within the 1st week
of life), and possibly congenital, that is, detected within the first
2months of life [6]. Other classifications take symptoms and time of
diagnosis into account and extend congenital tumor criteria to cover
the first year of life [6,7]. Although congenital growth does not
seem to be correlated with any particular tumor, glioblastoma,
and teratoma are most often diagnosed, with astrocytoma,
We describe the case a 2-day-old female with congenitalglioblastoma. Total resection was followed by adjuvant and highdose chemotherapy, as indicated by the current Italian infantprotocol. The child is alive and well 18 months after diagnosis. Areview of 67 selected congenital brain tumors showed the mortalityrate was 82%. Even though the majority of patients had glioblastoma,only 5/67 had received adjuvant therapy. To ensure optimal
outcomes, we recommend total or subtotal surgical resection,followed by adjuvant and high dose chemotherapy. Given the lackspecific protocols for congenital brain tumors an internationalconsensus seems to be needed, starting with congenital glioblas-toma. Pediatr Blood Cancer 2009;53:124–126.� 2009 Wiley-Liss, Inc.
Key words: chemotherapy; congenital brain tumors; high dose chemotherapy; infant
——————Additional Supporting Information may be found in the online version
of this article.
1Unit of Pediatric Oncology and Haematology, Ospedale ‘‘S. Maria
della Misericordia’’, A.O. di Perugia, Italy; 2Division of Neurosurgery,
Meyer Children’s Hospital, Florence, Italy
*Correspondence to: G.M. Milano, Division of Pediatric Oncology and
Haematology, Ospedale ‘‘S. Maria della Misericordia,’’ A.O. di
Perugia Sant’Andrea delle Fratte, 06156, Perugia, Italy.
E-mail: [email protected]
Received 27 December 2008; Accepted 10 February 2009
� 2009 Wiley-Liss, Inc.DOI 10.1002/pbc.22008Published online 23 March 2009 in Wiley InterScience(www.interscience.wiley.com)
124 Brief Reports