Daniela Di Martino

Prospective molecular monitoring of BCR/ABL transcript in children with Ph+ acute lymphoblastic leukaemia unravels differences in treatment response

Summary. Children with Philadelphia‐chromosome‐positive (Ph+) acute lymphoblastic leukaemia (ALL) represent a subgroup at very high risk for treatment failure, despite intensive chemotherapy. However, recent retrospective studies showed that Ph+ childhood ALL is a heterogeneous disease with regard to treatment response. We have prospectively monitored, by reverse transcription polymerase chain reaction (RT‐PCR) during follow‐up, the presence of the BCR/ABL fusion transcript in Ph+ ALL children diagnosed in the Italian multicentre Associazione Italiana Ematologia Oncologia Pediatrica ALL‐AIEOP‐95 therapy protocol. To our knowledge, this is the first report on the evaluation of minimal residual disease (MRD) in childhood Ph+ ALL prospectively enrolled in an intensive, Berlin–Frankfurt–Munster (BFM)‐type treatment protocol. Twenty‐seven of 36 (75·0%) Ph+ patients consecutively enrolled into the high‐risk group of the AIEOP‐ALL protocol between May 1995 and October 1999 were successfully analysed. Twenty were good responders to the pre‐phase of prednisone/intrathecal methotrexate treatment (PGR) and seven were poor responders (PPR). Within the PPR group, the RT‐PCR monitoring constantly showed positivity for the BCR/ABL fusion transcript and all the patients died of disease progression. In contrast, highly sensitive qualitative RT‐PCR monitoring revealed heterogeneity within the PGR group of Ph+ childhood ALL patients. Three different subgroups could be defined, according to the clearance of Ph+ cells within the first 5 months of treatment. This provides useful information on the capability of chemotherapy to reduce the leukaemic clone, with prognostic implications.

Unrelated hematopoietic stem cell transplantation for Cernunnos‐XLF deficiency

Abstract:  Cernunnos‐XLF deficiency is a rare CI characterized by a defective DNA DSB repair mechanism. Its clinical manifestations are growth retardation, dysmorphic features, malformations, and severe B‐ and T‐cell lymphopenia. BM failure may complicate the clinical picture. To date, there have been no described patients with CSy undergoing allogeneic HSCT. We report a case of CSy treated successfully with unrelated allogeneic HSCT after a reduced‐intensity conditioning regimen. Two yr after HSCT, the patient maintains full donor engraftment, normal hematopoiesis, and progressively improving immune competence, thus suggesting that HSCT may be the treatment of choice for CSy.

Long-lasting Hypogammaglobulinemia Following Rituximab Administration for Epstein-Barr Virus-Related Post-Transplant Lymphoproliferative Disease Preemptive Therapy

HIGH LEVELS of Epstein-Barr virus (EBV) DNA have been associated with the risk of developing post transplant lymphoproliferative disease (PTLD) after allogeneic bone marrow transplant (1), and rituximab has been reported to represent an effective preemptive therapy for this complication (2). In one study, B cell lymphopenia following rituximab administration has been reported persisting for a median of 9 months, with a range upper limit of 12 months (2). We report our experience with long-lasting hypogammaglobulinemia following rituximab administration for high-level EBV DNA in a 9-year-old boy. P.P. received an unrelated (matched for 5/6 antigens) allogeneic bone marrow transplant for marrow and testis relapsed acute lymphoblastic leukemia. Graft-versus-host disease (GVHD) prophylaxis was carried out with cyclosporin (CSA), three doses of rabbit anti-thymocyte globuline (ATG), and methotrexate. On day 117, due to the occurrence of a grade III skin and liver acute GVHD, he received a second course of ATG and high-dose methylprednisolone. With these treatments, clinical conditions improved. On day 1129 after transplant, while under immunosuppressive therapy with oral CSA and methylprednisolone, resulting EBV DNA levels were 10,000 GEq/105 mononucleated cells, with 6.4% CD191 and 3.6% CD201 lymphocytes in his peripheral blood, and increasing lactic dehydrogenase (LDH) levels (1280 IU/L). Therefore, rituximab at a dose of 3759 mg/m2 was administered. In the following days, EBV DNA levels dropped to undetectable values, CD191 and CD201 lymphocytes became 0%, and LDH levels decreased; therefore, no further dose of rituximab was administrated. A program of substitutive intravenous immunoglobulin administration was implemented because of rituximab-induced severe hypogammaglobulinemia. At present, 27 months after rituximab administration, the patient is still receiving substitutive intravenous immunoglobulins because of persistent hypogammaglobulinemia, with the following levels before administration: immunoglobulin G (IgG) 285 mg/dl (normal values 453–1350 mg/dl), IgA 55 mg/dl (normal values 19–146 mg/dl), and IgM 4 mg/dl (normal values 20–100 mg/dl). His CD201 lymphocytes are 2.6% (normal values for the age 5.8–15.8%) (3), but, surprisingly, the molecular analysis of IgM heavy-chain third complementary-determining region (HCDR3) expression by means of fingerprinting on peripheral blood mononucleated cells (4) showed a normal polyclonal expression of IgM HCDR3 for the two gene families, VH3 and VH6. His immunosuppressive therapy is now represented by micophenolate mofetil and low doses of methylprednisolone. Rituximab has been reported as effective preemptive therapy for EBV-induced PTLD following allogeneic bone marrow transplantation (BMT) (2). B cell lymphopenia following rituximab therapy has been reported to last for a median of 9 months, and a maximum range of 12 months (2), with severe infectious complications, especially in patients having extensive chronic GVHD. In our patient, severe hypogammaglobulinemia persists more than 2 years after rituximab infusion, with low levels of CD201 lymphocytes, but with a normal polyclonal expression of HCDR3 for VH3 and VH6 gene families,

An unusual pattern of B-cell immunological reconstitution after allogeneic stem cell transplantation: a possible correlation with CMV reactivation?

Abstract:  IR after HSCT is a slow process that involves several components of the immune response, and, in allogeneic setting, it can be delayed by GvHD and immuno‐suppressive therapy. Our study on IR post‐HSCT included a child with FA who underwent MUD transplantation. To evaluate B, T and NK cell reconstitution and to investigate the differentiation of B lymphocyte repertoire, this patient was carefully monitored at various time points by IgHCDR3 (third complementarity determining region of the immunoglobulin heavy chain) fingerprinting and by FACS analysis. IgHCDR3 fingerprinting showed a strong oligoclonality of IgM and IgG profiles from day +60 to +180 post‐transplant. CMV reactivation was present at the same time points and overlapped the clonal pattern shown in IgHCDR3 fingerprinting. Immunophenotype analysis showed early repopulation of T and NK cells following HSCT, whereas B cells increased first at one yr post‐transplant. The overlapping of virus reactivation and B‐cell clonal expansion seems to suggest that B lymphocytes may be involved in the CMV immunological response, at least in the early time points after HSCT when the immune repertoire is still reconstituting.

Allogeneic hematopoietic stem cell transplantation in congenital disorders: A single-center experience

Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is the treatment of choice for a variety of congenital disorders. We report the experience of children affected by congenital diseases other than bone marrow failure syndromes who received allo‐HSCT over a period of 25 years at G. Gaslini Paediatric Research Institute. HSCTs were performed in 57 children with congenital diseases (25 with congenital immunodeficiencies, 10 with severe combined immunodeficiencies, and 22 with metabolic diseases). Overall survival rate at 3 years in the whole group of patients was 76.9%, with a trend in favor of better outcome in children with metabolic diseases and in those who received cord blood cells (85.9%) vs bone marrow cells (72.4%).

Evaluation of Chimerism Dynamics after Allogeneic Hematopoietic Stem Cell Transplantation in Children with Nonmalignant Diseases

It is recognized that chimerism following hematopoietic stem cell transplantation (HSCT) is a dynamic process. The aims of this study were to describe the evolution of chimerism in children with nonmalignant diseases who underwent allogeneic HSCT, and to analyze the risk factors influencing chimerism status. A total of 101 HSCTs were performed in 85 patients with nonmalignant diseases. The donor was unrelated in 62.4% of HSCTs. Reduced-intensity conditioning (RIC) regimen was administered in 48.5% of patients. Acute graft-versus-host disease (aGVHD) occurred in 51.7% and chronic GVHD (cGVHD) in 39.7% of patients. Analysis of chimerism was performed through amplification of 9 specific short tandem repeats by polymerase chain reaction at engraftment and 1, 6, and 12 months after HSCT. Upon first evaluation, complete chimerism (CC) was detected in 34.7% and mixed chimerism (MC) in 55.4%, whereas graft failure occurred in 9.9% of patients. Severe aGVHD was associated with CC (P = .031). The last chimerism evaluation showed CC in 72.1%, stable MC in 12.8%, and progressive MC in 3.5%. CC was associated with a higher incidence of aGVHD (P = .016) and cGVHD (P = .022), whereas the RIC regimen was associated with graft failure (P = .026). One- and 3-year overall survival (OS) was 87.4% and 80.5%, respectively, with a lower OS at 3 years in patients with CC compared with those with MC (P = .008). aGVHD and cGVHD represent factors favoring CC, thus close, careful follow-up of chimerism is recommended in patients affected by nonmalignant disease.

233 PREFERENTIAL USAGE OF INCOMPLETE REARRANGEMENTS IN PRECURSOR B-ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL)

Southern blot analysis of T-cell receptor TCR genes rearrangements is usefull for diagnostic studies on the clonality of lymphoproliferative diseases and for a better assigment of the cell lineage of leukemic cells.We analyzed 26 cases of precursor B-ALL in aim to detect the presence of TCR-δ gene rearrangements. In fact lineage crossover of gene rearrangements have been osserved quite frequently in neoplastic cells. We found 6/26 patients in germline configuration, 5/26 deleted and 15/26 with at least one allele reananged. Taking into account previous studies (Breit et al. 1993) we were able to define exactly in 8/26 cases the gene segments involved in the rearrangement; DNA extracted from bone marrow samples was digested with Eco RI, Hind III and Bgl II restriction enzymes. The hybridization with TCRJδ 1 probe showed that the majority of the rearrangements were Vδ2Dδ3 and Dδ2Dδ3.It is possible that the crossover rearrangements reflect the earliest steps of recombinational event in rearranging antigen-receptor gene loci. Besides the Vδ2Dδ3 rearrangement, the most frequently observed in precursor B-ALL, may be the earliest recombinational event in TCR-δ gene during normal T-cell ontogeny. Amplification by PCR technique, using specific primers revealed the presence of a specific band of leukemic clone and in one case we found the sequence of junctional region between Vδ2 and Dδ3 gene segments. In this particular case, the insertion of 6 nucleotides creates a unique clonal marker of leukemic cells that can be used for the detection of minimal residual disease (MRD).