Immune tolerance therapy for haemophilia.

The development of anti-factor VIII and anti-factor IX allo-antibodies in haemophilia A and B, respectively, remains a serious complication of treatment for these two X-linked haemostatic disorders, with major clinical and economic consequences. Treatment of this potentially fatal complication remains one of the greatest challenges facing haematologists at the beginning of the 21st century. Immune tolerance induction (ITI) therapy has been generally accepted as the best available treatment, extinguishing the inhibitor and permitting a resumption of standard dosing schedules. Although there have been several established protocols for ITI therapy developed over the last quarter century, the optimal scheme in terms of safety, clinical efficacy and pharmacoeconomic considerations has yet to be determined.

Analysis of clonal rearrangements of the Ig heavy chain locus in acute leukemia.

Clonal rearrangements of the Ig heavy chain (IGH) locus occur in nearly all cases of B-cell precursor acute leukemia (BCP-ALL). Some of these rearrangements may be detected by polymerase chain reaction (PCR) using VH gene framework III (FRIII) and JH consensus primers. However, about 20% of BCP-ALLs fail to amplify with this technique. To determine the causes of these PCR failures and to investigate any possible association with specific subgroups of disease, we analyzed 72 acute leukemias of defined immunophenotype and cytogenetics, comparing FRIII with VH-family leader-specific PCR methods and Southern blotting. Of 37 BCP-ALL cases, 6 (16.2%) failed totally to amplify with FRIII and JH primers. None of these cases amplified with VH leader primers. Additionally, all cases retained germline VH6 genes and 5 of 11 rearranged alleles amplified with a consensus DH primer, indicating that these rearrangements represented biallelic DH-JH recombinations. Among the 6 FRIII and VH leader PCR-negative BCP-ALL cases, there was no common immunophenotype or consistent cytogenetic abnormality, although all showed structural chromosomal abnormalities and 3 of 5 successfully karyotyped had abnormalities of chromosome 12p. 13 cases with t(9;22)(q34;q11) Philadelphia chromosome-positive [Ph+]) and IGH rearrangements (9 BCP-ALL and 4 biphenotypic cases) were also analyzed. Of 23 rearranged IGH alleles, 19 (82%) were positive by FRIII PCR, and all 4 remaining alleles were amplified by VH leader primers. Use of the leader primers in these Ph+ cases also detected 3 additional clonal rearrangements that were not anticipated from Southern blotting; such unexpected bands were not observed in 21 other Ph- cases. The additional bands represented "new" and unrelated VH rearrangements rather than VH-VH replacement events. We conclude that biallelic DHJH rearrangements occur in a subgroup of BCP-ALL; in these cases, the activation of the full VHDHJH recombination mechanism had not occurred. Therefore, these cases of BCP-ALL were arrested at an early stage of B-cell differentiation. In contrast, all Ph+ BCP-ALLs and biphenotypic acute leukemias, which may represent the transformation of multipotent hemopoietic stem cells, had undergone VHDHJH recombination. Of 9 Ph+ BCP-ALL cases, 3 also showed ongoing VHDHJH rearrangement, reflecting the persistent expression of the VHDHJH recombinase. Finally, sequence analysis of 33 rearranged VHDHJH genes showed that only 3 including 2 Ph+ BCP-ALL maintained an intact open-reading frame. Loss of the open-reading frame occurred not only because of out-of-frame VHDH and DHJH joining, but also because of VH gene mutation and deletion. These data show that most BCP-ALLs may represent the neoplastic transformation of BCPs destined to die in the bone marrow.

Deletions and rearrangement of CDKN2 in lymphoid malignancy.

Recurrent abnormalities of the short arm of chromosome 9, including translocations and interstitial deletions, have been reported in both leukemia and lymphoma. The pathologic consequences of these abnormalities remain unknown. The cyclin-dependent kinase 4 inhibitor (CDKN2) gene, which maps to 9p21, has been implicated by the finding of a high frequency of biallelic deletions in leukemic cell lines. We have determined the incidence of structural abnormalities affecting CDKN2 by DNA blot in a panel of 231 cases of leukemia and lymphoma and 66 cell lines derived from patients with lymphoid malignancies with defined cytogenetic abnormalities. Structural alterations of CDKN2 were seen in 20 (8.3%) of all fresh cases and 10 (15.1%) of all cell lines. Biallelic CDKN2 deletions were seen in 11 of 53 (21%) cases of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There was no association with any particular cytogenetic abnormality. Biallelic deletions were also found in high-grade and transformed non-Hodgkin's lymphoma (NHL) of both B- and T-cell lineages. In two cases of transformed NHL, analysis of sequential samples showed loss of CDKN2 with transformation. Neither deletions nor rearrangements of the CDKN2 gene were seen in any of the 119 leukemias of mature B or T cells analyzed. Biallelic deletions of CDKN2 were observed in 6 of 13 NHL cell lines. Three of the 6 cases had undergone transformation from low- to high-grade disease: in 2 of these cases it was possible to show that the CDKN2 deletions were present in fresh material from the patient and were therefore not an artifact of in vitro culture. Rearrangements of CDKN2 were seen in 2 cases (4%) of BCP-ALL, in 1 case of B-NHL, and in 1 Burkitt's lymphoma cell line and suggest the presence of a "hot spot" for recombination in the vicinity of the CDKN2 gene. These data indicate that the loss of CDKN2 expression may be involved in the pathogenesis of a subset of BCP-ALL, some high-grade NHL, and in the transformation of NHL from low- to high-grade disease. CDKN2 deletions and rearrangements occurred in the absence of detectable cytogenetic changes of chromosome 9p in 25 of 30 (83%) cases. Finally, of 10 cases of BCP-ALL that produced overt, transplantable leukemia in mice with severe combined immunodeficiency (SCID), seven showed biallelic CDKN2 deletions. In contrast, none of 11 cases that failed to engraft showed biallelic CDKN2 deletions. BCP-ALL cases that lack CDKN2 expression may have a particular propensity to grow in SCID mice.

Acute myelomonocytic leukemia with t(10;11)(p13;q23): heterogeneity of breakpoints at 11q23 and association with recombinase activation.

The human trithorax homolog gene (MLL) is directly involved in over 90% of cases of acute leukemia with abnormalities of 11q23. However, involvement of other genes at 11q23 both centromeric and telomeric of MLL has been identified in different subtypes of leukemia and lymphoma. We describe a case of acute myelomonocytic leukemia (AMML; FAB type M4) with t(10;11)(p13;q23) in which the breakpoint at 11q23 was centromeric to the MLL gene and distinct from the breakpoint seen in promyelocytic leukemias with t(11;17)(q23;q22), thus providing further evidence of heterogeneity of breakpoints in 11q23 in acute leukemia. Rearrangements of immunoglobulin (IG) and T-cell receptor (TCR) genes were also observed, with no immunophenotypic evidence for commitment to the lymphoid lineages, indicating that inappropriate activation of the recombinases may be a feature of this particular variant translocation.

Tumour lysis following hydrocortisone prior to a blood product transfusion in T-cell acute lymphoblastic leukaemia.

The acute tumour lysis syndrome is a well recognised complication of chemotherapy for lymphoid malignancies. There are few reports, however, of this complication after corticosteroid therapy alone. We report a case of T-cell acute lymphoblastic leukaemia who developed the biochemical picture of tumour lysis after two doses of hydrocortisone given prior to platelet transfusion. Prophylactic corticosteroids prior to blood product infusion should be reserved for patients who have experienced febrile or allergic reactions in the past and it is suggested that they should only be administered to patients with active lymphoid malignancies with due caution.

Disturbances in T-cell subpopulations in B-cell chronic lymphocytic leukaemia.

The relative proportions of immunoregulatory T-cell subpopulations defined by OKT monoclonal antibodies are disturbed in chronic lymphocytic leukaemia (CLL) patients (even early mild cases) compared with normal subjects in the same age range. The mean ratio OKT4+:OKT8+ (helper:suppressor/cytotoxic) is reversed from the normal 1.6:1 to 1:1.9. The absolute concentration of each OKT population in the circulation is slightly higher than normal, the increase in OKT8+ being the most significant. The E-rosette-forming cells did not always correlate with OKT+ cells and, in four cases, the discrepancy between the size of the OKT3+ population and the sum of OKT4+ and OKT8+ suggested the presence of T cells with an immature (thymic) phenotype in peripheral blood. These abnormalities may account for the depressed immune function of CLL patients.