Int J Hematol (2014) 99:208–212 DOI 10.1007/s12185-013-1489-3
CASE REPORT
Different outcomes of allogeneic hematopoietic stem cell transplant in a pair of twins affected by juvenile myelomonocytic leukemia Simone Cesaro • Paola De Filippi • Annamaria Di Meglio • Anna Leszl • Svetlana Donska • Ada Zaccaron • Claudia Cagioni • Roberta Galavotti • Cesare Danesino • Fiorenza Aprili • Chiara Cugno • Geertruy te Kronnie Marco Zecca • Silvia Bresolin
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Received: 14 August 2013 / Revised: 27 November 2013 / Accepted: 27 November 2013 / Published online: 12 December 2013 Ó The Japanese Society of Hematology 2013
Abstract A twin pair affected by juvenile myelomonocytic leukemia (JMML) with the same somatic PTPN11 mutation and abnormal chromosome 7 in bone marrow samples but distinct prognostic gene expression signatures, received a matched-unrelated donor and matched-unrelated cord blood transplant, respectively. Both twins fully engrafted, but after 6 months, the twin with an acutemyeloid-like (AML-like) signature at diagnosis rejected the graft and had an autologous reconstitution. A second transplant with an unrelated 5/6-HLA-matched-loci cord blood performed after 4 months from rejection was unsuccessful. After 25 months from diagnosis, the twin with the AML-like gene expression signature died of liver failure while on progression of his JMML. The other twin, who had a non-acute-myeloid-like (non-AML-like) gene expression signature at diagnosis is in complete hematological remission with full donor chimera. This observation Electronic supplementary material The online version of this article (doi:10.1007/s12185-013-1489-3) contains supplementary material, which is available to authorized users. S. Cesaro (&) A. Zaccaron C. Cugno Pediatric Hematology Oncology, Azienda Ospedaliera Universitaria Integrata, Piazzale L.A. Scuro, 10 Policlinico G.B. Rossi, 37134 Verona, Italy e-mail:
[email protected] P. De Filippi C. Cagioni C. Danesino Medical Genetics, Department of Molecular Medicine, University of Pavia, Pavia, Italy A. Di Meglio A. Leszl G. te Kronnie S. Bresolin Department of Women and Child Health, University of Padova, Padua, Italy S. Donska Pediatric Hematology Oncology, Children Hematology Center, Okhmatdet Hospital, Kiev, Ukraine
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suggests a biological diversity of JMML also in patients with a common genetic background. Keywords Juvenile myelomonocytic leukemia Twins Hematopoietic stem cell transplant Gene expression-based diagnostic classification
Introduction Juvenile myelomonocytic leukemia (JMML) is a rare clonal disease of hematopoietic stem cells that represents about 2–3 % of pediatric malignancies and has an incidence of 1.2 case/million people [1–3]. JMML has a typical clinical picture at presentation characterized by splenomegaly, hepatomegaly, lymphadenopathy, and skin rash. The typical blood count findings are leukocytosis, monocytosis, anemia, thrombocytopenia often with dysplastic features of one or more cell lineages. Monosomy 7 is the cytogenetic abnormality reported in about 25 % of patients. R. Galavotti Department of Life Science and Reproduction, University of Verona, Verona, Italy F. Aprili Cytogenetics, Department of Pathology and Diagnostics, Azienda Ospedaliera Universitaria Integrata, Verona, Italy M. Zecca Pediatric Hematology Oncology, Department of Pediatrics, IRCS San Matteo, Pavia, Italy
Stem cell transplant for JMML in twins
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Mutations affecting RAS signaling pathway were found in 90 % of JMML patients including the genes for RAS (25 %), PTPN11 (35 %), NF1 (10–15 %) and c-CBL (15 %) [4]. The disease is aggressive and patients do not usually survive beyond the first year from diagnosis due to hemorrhage and infection. At present the only effective treatment is hematopoietic stem cell transplantation (HSCT), but rejection and relapse are still significant and frequent causes of treatment failure in about 50 % of cases [5]. A recent retrospective study that used a gene expression profile (GEP) classification algorithm identified 2 groups of JMML patients with different transplant outcomes. Moreover, from the multivariate analysis of clinical and biological variables including age, HbF level, and platelet count, only the gene expression-based classification was significant in predicting patient outcome [6]. In this short report, we describe the transplant outcomes of a pair of twins affected by congenital JMML.
Case report The twins were born at 36th week of gestation from a monochorionic, monoamniotic pregnancy. The pregnancy presented a threatening of premature labor at the 28th week
of gestation. At 19 months of age, one of the twins presented with fever, pallor, hepatosplenomegaly, lymphadenopathy and skin rash. The subsequent blood and bone marrow exams were consistent with JMML. While more laboratory data were requested for the confirmation of JMML in Twin_01, the high suspicion of JMML triggered the assessment of blood and marrow in the second twin (Twin_02) that was also consistent with a diagnosis of JMML. The hematological, cytogenetic, molecular characteristics are shown in Table 1. At diagnosis, the twins presented the same missense mutation at PTPN11E76K and a rare 46, XY, -7 ?mar karyotype (Fig. 1a, b). FISH analysis defined the monosomy 7 with marker chromosome as a deletion of the short and long arms of chromosome 7 with a remaining part of chromosome 7 close to the centromeric region (Fig. 1b). Array CGH delineated diploid chromosome 7, p12.1 harboring sequences of 3 genes and known noncoding RNAs in each of the twins (Supplemental Figure 1). The presence of the same rare aberration of der7 of maternal origin (Supplemental Figure 2) along with the same mutations of PTPN11 strongly corroborates the hypothesis that both events occurred in utero in one twin fetus first, the aberrations later during fetal life being transmitted to the co-twin via the common placenta. Even though Noonan syndrome phenotypical features were
Table 1 Main characteristics of the twins at diagnosis of JMML Twin_01
Twin_02
Sex
Male
Male
Age at diagnosis (years)
19 months
19 months
Symptoms/signs
Fever, hepatomegaly, splenomegaly, lymphadenopathy, skin rash
No symptoms, diagnosis started 3 weeks after diagnosis of Twin_01
Spleen size below costal margin (cm)
8
1
Leukocyte count at diagnosis 9109/L
12.8
10.6
9
Monocyte count at diagnosis 910 /L
5.88
2.01
Platelet count at diagnosis, 9109/L
56 we have in our records 32
56 and 55
Hemoglobin level (g/dL)
8.2
8.2
Percentage of HbF at diagnosis
7.4 (normal for age)
10 (normal for age)
Percentage of blasts, peripheral blood
3%
13.6 %
Bone marrow dysplasia
Trilineage
Trilineage
Cytogenetic
45, XY, -7 [1]/46, XY, der [6]/46, YX [1]
45, XY, -7 [15]/46XY, der [3]/46, XY [3]
Oncogene mutations (on peripheral blood and bone marrow)
PTPN11, c.227A[G exon 3 (p.Glu76Gly)
PTPN11, c.227A[G exon 3 (p.Glu76Gly)
GEP-based diagnostic classification
AML-like
Non-AML-like
Treatment before HSCT
Prednisone (2 mg/kg/day) and cyclosporin (5 mg/kg/day) for 1 month
Prednisone (2 mg/kg/day) for 1 month Isotretinoin, 6-mercaptopurine for 7 months
Isotretinoin, 6-mercaptopurine for 4 months 59 cytarabine 40 mg/m2 Interval between diagnosis and HSCT
8 months
1 year
GEP genome expression profile, AML acute myeloid leukemia, HSCT hematopoietic stem cell transplantation
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Fig. 1 Overview molecular features at diagnosis in both twins. a Sanger sequencing analysis. Sanger sequencing was used as first screening to detect PTPN11 mutations in the bone marrow of both twins. Asterisks show the nucleotide involved in the mutation in exon3 of PTPN11 (c.227A[G, E76K). b Cytogenetic and FISH analysis of BM samples. Karyotype analysis using Q-banding
revealed the presence of monosomy 7 and a marker chromosome of unknown origin. FISH analysis using probe CEP 7 Spectrum Orange allowed identification of the mar chromosome as der7. In the metaphase, one normal chromosome 7 and one ‘‘der 7’’ (smaller signal) are detected, as indicated by arrows. Signals of normal chromosome 7 and der7 can also be observed in the interphase nuclei
absent in the twins, we analyzed other tissues looking for PTPN11 mutations in order to rule out the hypothesis that the genomic aberrations in the hematopoietic cells may have occurred also in the common mesentoderm progenitor cells. We analyzed fibroblasts and hair follicles to fully exclude the presence of mutations beyond the hematopoietic compartment. No mutation of PTPN11 was detected in either fibroblasts or hair bulbs. Also two copies of chromosome 7 were found in fibroblasts and hair bulbs of the twin pair. Moreover, sequence analysis of SETBP1 and JAK3, genes recently related with a poor outcome in JMML patients, revealed the absence of mutations in both Twin_01 and Twin_02. At the conclusion of the diagnostic workup, two main differences were noted in the twin pair: the symptoms at presentation, especially the important hepatosplenomegaly in Twin_01, and distinct GEP signatures— the diagnostic classifier revealed an AML-like signature in Twin_01 and a non-AML-like signature in Twin_02 (Table 1; Supplemental Appendix for method of Diagnostic Classifier). In the absence of HLA-matched related donors, a search for unrelated donors or cord blood units was opened for both twins at 4 months after diagnosis. Two unrelated donors were identified within 4 and 6 months from opening the search, and both twins proceeded with HSCT, general clinical conditions being good and underlying JMML well-controlled. Table 2 shows the main characteristics of the two transplants. The twins engrafted
rapidly both for polymorphonuclear cells and platelets. The post-transplant outcome was characterized by a grade II acute graft versus host disease (GVHD) in Twin_01 who responded to a standard dose of 2 mg/kg of prednisone. No chronic GVHD occurred. The monthly check of donor/ recipient chimera on peripheral blood (and on bone marrow every 3 months) by very short tandem repeats test on DNA showed a full donor chimera for both twins until day ?180. After that, Twin_01 completely rejected the graft despite immediate withdrawal of immunosuppressive treatment whilst Twin_02 continued to show a full donor chimera. The treatment with isotretinoin and 6-mercaptopurine was resumed for Twin_01 when autologous reconstitution with leucocytosis and monocytosis became apparent and a second search for an unrelated donor or cord blood unit was started. Within 4 months a cord blood unit that mismatched at allelic level locus A, but matched on B, C and DRB1 loci was identified. Twin_01 underwent a second HSCT 3 months after rejection and 11 months from the first HSCT. The conditioning regimen was thiotepa 10 mg/kg, fludarabine 150 mg/m2, etoposide 60 mg/kg, and serum anti-thymocyte serum (FreseniusÒ) 10 mg/kg. Prophylaxis of GVHD was with cyclosporin 3–5 mg/kg and prednisone 1 mg/kg for 4 weeks. Twin_01 received 7.2 9 107/kg of total nucleated cells and 1.02 9 106/kg of CD34? cells. Apart from fever of unidentified origin, the post-transplant outcome was smooth and a polymorphonuclear engraftment
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Table 2 Main transplant characteristics of twins with JMML Twin_01
Twin_02
Weight (kg)
10.5
12.8
Age at transplant (years)
2.3
2.6
Donor source
Unrelated, bone marrow
Unrelated, cord blood
D/R loci HLA-matched (at allelic level)
A, B, C, DRB1, DQB1
A, B, C, DRB1, DQB1
D/R blood group
0?/A?
A-/A?
D/R CMV sero status
pos/neg
neg/neg
Conditioning regimen
i.v. Busulfan, 19.2/mg/kg Cyclophosphamide, 120 mg/kg
i.v. Busulfan, 19.2/mg/kg Cyclophosphamide, 120 mg/kg
Melphalan, 140 mg/m2
Melphalan, 140 mg/m2
Rabbit anti-thymocyte globulin, 11.25 mg/kg
Rabbit anti-thymocyte globulin, 7.5 mg/kg
Ciclosporin 3 mg/kg
Ciclosporin 3 mg/kg
Short methotrexate (day ?1, ?3, ?6)
Prednisone 1 mg/kg (from day -1 to day ?30)
5 9 108//kg
8.1 9 107/kg
GVHD prophylaxis
TNC infused
6
CD34? cells infused
3.5 9 10 /kg
0.6 9 106/kg
Neutrophil engraftment ([0.5 9 109/L)
Day ?27
Day ?24
Platelet engraftment ([50 9 109/L)
Day ?28
Day ?35
Acute GVHD
I
0
Chronic GVHD
No
No
Chimerism
Full donor chimera from day ?30 to day ?180, then rejection
Full donor chimerism
Autologous reconstitution
Day ?200
No
Status at last follow-up
Died of liver failure while on progression of JMML
Complete hematological remission Off every immunosuppressive treatment
D/R donor/recipient, HLA human leucocyte antigen, TNC total nucleated cells, GVHD graft versus host reaction
occurred at day ?10. The first control of chimerism on bone marrow at day 22 showed 20 % of recipient cells in the presence of dysplastic monocytes. Following the suggestions of a previous report [7], the ongoing immunosuppressive treatment (cyclosporin, low-dose prednisone) was withdrawn with the aim to reverse the graft rejection by inducing an acute GVHD reaction. As result of this, the patient had a grade III GVHD (skin, gut) that responded to a 2-week course of low dose of steroids (1 mg/kg/day). After withdrawal of immunosuppression treatment Twin_01 presented a progressive increase of white blood cells and dysplastic monocytes that was controlled with the resumption of chemotherapy, low dose of oral 6-mercaptopurine, cytarabine and retinoic acid. Further controls of marrow chimerism after 60, 90, 120 days from the second HSCT showed a progressive increase of recipient cells to 80 %. Sequence analysis of SETBP1 and JAK3 genes excluded the presence of mutations in the bone marrow cells after relapse. After 25 months from diagnosis, Twin_01 died of liver failure while on progression of disease whilst Twin_02, 4.5 years old, is alive and well with full donor chimerism and no sign of JMML.
Discussion This report describes the very rare event of a pair of twins affected by JMML. No other similar cases have been published so far, except 2 twins affected by chronic myelomonocytic leukemia with t(7;16) translocation [8]. The coincidence of cancer in twins has been demonstrated epidemiologically only for monozygotic twins with acute leukemia and non-Hodgkin lymphoma [9]. The studies of leukemia in twins suggests that the origin of the major part of pediatric leukemias has a 2-stage development, one in utero, during fetal hematopoiesis, that originates a preleukemic clone, followed by a complementary or secondary genetic event, usually during the post-natal period and with different times of latency, that determines the overt leukemia [10]. In this case of monochorionic twins, an alternative possibility is that both leukemogenetic events, monosomy 7/der7 of maternal origin and PTPN11 mutations occurred both at an early stage of fetal life in hematopoietic precursors of one of the twins and passed subsequently, possibly as a slightly more differentiated hematopoietic clone, to the co-twin by common placental
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circulation. The second peculiar finding is the different outcomes of HSCT. Despite of the occurrence of JMML in the two monozygotic twins with the same molecular and cytogenetic characteristics, Twin_01 rejected 2 unrelated HSCTs in spite of myeloablative conditioning regimens, whereas in Twin_02 the HSCT was successful. In the largest study published so far on the outcome of HSCT in JMML, only age [4 years and female sex were significantly associated with lower event-free survival [5]. None of these characteristics were present in our twins, so we attribute the observed different outcomes to other features. The highly discordant clinical disease course in the twins is in close concordance with our previously published results on 44 cases with JMML. This study shows that patients with an AML-like signature have a poor prognosis, whereas patients with a non-AML-like signature have a favorable prognosis following HSCT [6]. The co-occurrence of monosomy 7 and PTPN11 mutation in the twins with an AML-like and a non-AML-like signature was also previously found to be present in other JMML patients with distinct GEP-based classification [6]. As described at diagnosis, the BM of Twin_01 presented with the same PTPN11E67K mutation, as well as the loss of the long and short arms of chromosome 7, der7, p12.1 and an AML-like GEP signature, also at relapse. A recent publication Sakaguchi et al. [11] reported a high incidence of SETBP1 and JAK3 mutations is JMML patients. The incidence was particularly relevant in PTPN11-mutated JMML patients with 9 out of 39 patients mutated for either SETBP1 or JAK3 or both these mutations. However, only 1 out of 8 patients with monosomy 7 had a secondary mutation in SETBP1 or JAK3. Altogether the study reported a paucity of gene mutations apart from the RAS pathway mutations in JMML BM samples. The absence of mutations in SETBP1 and JAK3 in the twin pair at diagnosis and also at relapses of Twin_01 indicated that the progression of the disease in Twin_01 cannot be attributed to these secondary mutations. To explain the high incidence of relapses after HSCT in JMML, we speculate that in relapsing patients leukemic progenitor cell clones are particularly resistant to myeloablative agents used in the conditioning regimens and give rise to the re-occurence of the disease with the same molecular and karyotype features. In conclusion, the prospective observation of the discordant outcomes of the two twins confirmed the prognostic relevance of the gene expression-based classification
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of JMML patients at diagnosis and should stimulate research into new strategies of treatment for a subgroup of patients in which the result of HSCT is not satisfactory. Acknowledgments The work was support by Associazione Italiana per la Ricerca sul Cancro (AIRC) to S.B., PRIN and Fondazione Cariplo to G.teK. We thank Lifeline Charity Trust for the support to the family and Anna Pegoraro, M.D., for the help editing the manuscript. Conflict of interest of interest.
The authors declare that they have no conflict
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