Martin L. Brecher

DNA Vaccine Expressing the Mimotope of GD2 Ganglioside Induces Protective GD2 Cross-reactive Antibody Responses

The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside.

Intermediate-dose methotrexate versus cranial irradiation in childhood acute lymphoblastic leukemia: a ten-year follow-up.

The cure rate of childhood acute lymphoblastic leukemia (ALL) has improved dramatically. Still there is a paucity of long-term data. With the improving cure rate, the quality of life and avoidance of second cancers have become important concerns. We evaluated 596 children and adolescents with ALL on Cancer and Leukemia Group B 7611 (CALGB 7611) who were randomized between 1976 and 1979 to receive intermediate-dose methotrexate (IDM) plus intrathecal methotrexate (IT MTX) or cranial radiation (CRT) plus IT MTX. After 10 additional years of follow-up, the pattern and significance of the results reported in 1983 are confirmed. IDM offered better hematologic protection (P < 0.0006), better testicular protection (P = 0.002), but CRT offered better central nervous system (CNS) protection (P < 0.0001). The retrieval rate for the 231 patients who relapsed while on therapy or within 6 months of elective cessation of therapy is 20 +/- 5%. For the 33 patients who relapsed more than 6 months after cessation of therapy, the retrieval rate is 49 +/- 10%. For all patients, the 12-year event-free survival was 37 +/- 3.6% and the overall survival was 49 +/- 3.5%. There were two cases of second malignancies reported in 3,502 person-years of survival. Both occurred following salvage therapy. There was no evidence of an excessive number of second primaries over the general population of children. There were no reported instances of clinical cardiopathy. After a median follow-up of 11 years, there have been no reports of cardiopathy and no evidence of an increased risk of second cancers in children treated on CALGB 7611. While the overall outcome is not what would be expected with modern therapy, one can conclude that CRT offered better CNS protection, but IDM offered better systemic and testicular protection. A small risk of second cancers or cardiac dys-function may be acceptable with therapies which produce long-term documented survival benefits.

Neurocognitive and neuroradiologic central nervous system late effects in children treated on pediatric oncology group (POG) P9605 (Standard Risk) and P9201 (Lesser Risk) acute lymphoblastic leukemia protocols (ACCL0131): A methotrexate consequence? A report from the children's oncology group

Concerns about long-term methotrexate (MTX) neurotoxicity in the 1990s led to modifications in intrathecal (IT) therapy, leucovorin rescue, and frequency of systemic MTX administration in children with acute lymphoblastic leukemia. In this study, neurocognitive outcomes and neuroradiologic evidence of leukoencephalopathy were compared in children treated with intense central nervous system (CNS)-directed therapy (P9605) versus those receiving fewer CNS-directed treatment days during intensive consolidation (P9201). A total of 66 children from 16 Pediatric Oncology Group institutions with “standard-risk” acute lymphoblastic leukemia, 1.00 to 9.99 years at diagnosis, without evidence of CNS leukemia at diagnosis were enrolled on ACCL0131: 28 from P9201 and 38 from P9605. Magnetic resonance imaging scans and standard neuropsychological tests were performed ≥2.6 years after the end of treatment. Significantly more P9605 patients developed leukoencephalopathy compared with P9201 patients (68%, 95% confidence interval 49%-83% vs. 22%, 95% confidence interval 5%-44%; P=0.001) identified as late as 7.7 years after the end of treatment. Overall, 40% of patients scored <85 on either Verbal or Performance IQ. Children on both studies had significant attention problems, but P9605 children scored below average on more neurocognitive measures than those treated on P9201 (82%, 14/17 measures vs. 24%, 4/17 measures). This supports ongoing concerns about intensive MTX exposure as a major contributor to CNS late effects.

Translocation t(1;22) in congenital acute megakaryocytic leukemia

Acquired chromosomal abnormalities have been reported in 80 patients with congenital acute leukemia, the commonest being t(4;11). We report here a case of acute megakaryocytic leukemia with a rare translocation of t(1;22)(p13.3;q13.3). The course of the disease was short, with the patient surviving less than a year after the initial diagnosis.

Profile of immunoglobulin heavy chain variable gene repertoires and highly selective detection of malignant clonotypes in acute lymphoblastic leukemia.

The predominant B cell immunoglobulin heavy chain variable gene (IgH‐V) usage and the uniquely rearranged, clonotype‐specific variable‐diversity‐joining region gene (VDJ) sequences were identified in patients with B cell acute lymphoblastic leukemia (B‐ALL) using a novel DNA‐based gene amplification strategy. The approach allows a thorough and sensitive determination of the number of clonal leukemic IgH rearrangements and their precise V gene usage. This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease‐free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones. An initial primary polymerase chain reaction (PCR), directed by an IgH‐J generic primer and a complement of family‐specific IgH‐V primers, defined the major B cell IgH‐V gene usage. Use of an IgH‐J generic primer supplanted the use of a constant region primer anchor and thus eliminated the need to target mRNA by the traditional RNA reverse transcription–PCR amplification method. Monoclonality of rearranged VDJ bands was further substantiated by high‐resolution denaturant gel electrophoretic analysis. The predominant amplified bands were subcloned and sequenced. By sequencing through VDJ juxtaposed regions, that is, the third complementarity‐determining region, clonotype‐specific primers were developed and used in a secondary clonotype primer‐directed PCR (CPD‐PCR) to detect, with extreme sensitivity and specificity, a unique B cell clone. Analysis of the products of the CPD‐PCR permitted the detection of a single malignant cell among 1 million polyclonal cells and superseded the constraints of prior studies that have provided a limited evaluation of family variable gene repertoire usage. Leukemic clonal rearrangements were detected in 100% of the eight cases of pediatric and two cases of adult B‐ALL studied. Two or more clonal IgH‐VDJ amplified sequences were observed in 50% of the B‐ALL bone marrows analyzed. In two cases, clonotype‐specific oligodeoxynucleotide primers, derived from B‐ALL VDJ sequences, directed the secondary CPD‐PCR, and disease activity was monitored after chemotherapy and allogeneic bone marrow transplantation. J. Leukoc. Biol. 57: 856–864; 1995.

Ewing sarcoma of the mandibular condyle: Multidisciplinary management optimizes outcome

Ewing sarcoma (ES) is a rare, primary malignancy of bone that occurs in childhood and early adolescence. Improved methods of diagnosis and treatment have dramatically increased survival over the last 20 years. Treatment mainstays are chemotherapy and surgical tumor resection. ES usually occurs in long bones of the axial skeleton; however, it may rarely arise in facial structures, particularly the mandible. In these cases, resection presents a challenging postsurgical reconstruction.

Induction of protective immune responses against NXS2 neuroblastoma challenge in mice by immunotherapy with GD2 mimotope vaccine and IL-15 and IL-21 gene delivery

The GD2 ganglioside expressed on neuroectodermal tumor cells is weakly immunogenic in tumor-bearing patients and induces predominantly IgM antibody responses in the immunized host. Using a syngeneic mouse challenge model with GD2-expressing NXS2 neuroblastoma, we investigated novel strategies for augmenting the effector function of GD2-specific antibody responses induced by a mimotope vaccine. We demonstrated that immunization of A/J mice with DNA vaccine expressing the 47-LDA mimotope of GD2 in combination with IL-15 and IL-21 genes enhanced the induction of GD2 cross-reactive IgG2 antibody responses that exhibited cytolytic activity against NXS2 cells. The combined immunization regimen delivered 1xa0day after tumor challenge inhibited subcutaneous (s.c.) growth of NXS2 neuroblastoma in A/J mice. The vaccine efficacy was reduced after depletion of NK cells as well as CD4+ and CD8+ T lymphocytes suggesting involvement of innate and adaptive immune responses in mediating the antitumor activity in vivo. CD8+ T cells isolated from the immunized and cured mice were cytotoxic against syngeneic neuroblastoma cells but not against allogeneic EL4 lymphoma, and exhibited antitumor activity after adoptive transfer in NXS2-challenged mice. We also demonstrated that coimmunization of NXS2-challenged mice with the IL-15 and IL-21 gene combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in inhibiting tumor growth. This study is the first demonstration that the mimotope vaccine of a weakly immunogenic carbohydrate antigen in combination with plasmid-derived IL-15 and IL-21 cytokines induces both innate and adaptive arms of the immune system leading to the generation of effective protection against neuroblastoma challenge.

Precursor B lymphoblastic leukemia with surface light chain immunoglobulin restriction: a report of 15 patients.

We describe 15 patients (9 children) with precursor B-cell (pB) acute lymphoblastic leukemia (ALL) with surface immunoglobulin (sIg) light chain restriction revealed by flow cytometric immunophenotyping (FCI). The same sIg+ immunophenotype was present at diagnosis and in 3 relapses in 1 patient. In 15 patients, blasts were CD19+CD10+ (bright coexpression) in 14, CD34+ in 12, surface κ+ in 12, surface λ+ in 3; in 8 of 8, terminal deoxyribonucleotidyl transferase (TdT)+; and in 4, surface IgD+ in 2 and surface IgM+ in 1. The 3 CD34– cases included 1 TdT+ case, 1 with t(1;19)(q23;p13), and 1 infant with 70% marrow blasts. One adult had CD10–CD19+CD20–CD22+CD34+ TdT+sIg+ blasts with t(2;11)(p21;q23). Blasts were L1 or L2 in all cases (French-American-British classification). Karyotypic analysis in 12 of 12 analyzable cases was negative for 8q24 (myc) translocation. Karyotypic abnormalities, confirmed by fluorescence in situ hybridization in 6 cases, included hyperdiploidy, t(1;19)(q23;p13), t(12;21)(p13;q22), t(9;22)(q34;q11), t(2;11)(p21;q23), and trisomy 12. The sIg light chain restriction in pB ALL might be present in neoplasms arising from the early, intermediate, and late stages of precursor B-cell maturation; sIg light chain restriction revealed by FCI does not necessarily indicate a mature B-cell phenotype, further emphasizing the importance of a multidisciplinary approach to diagnosing B-lymphoid neoplasms.

Extra copies of chromosome 2 are a recurring aberration in ALK-negative lymphomas with anaplastic morphology

The purpose of this study was to evaluate fluorescence in situ hybridization abnormalities of the 2p23 anaplastic lymphoma kinase (ALK) gene loci in lymphomas with anaplastic morphology. We studied 24 anaplastic large cell lymphomas (ALCL) classified by World Health Organization criteria [17 primary nodal/systemic (10 ALK+, 7 ALK−), seven primary cutaneous], and 17 additional non-Hodgkins lymphomas [one ALK+ B-lineage lymphoma, 14 ALK− diffuse large B-cell lymphomas (seven anaplastic variants, five nonanaplastic, two secondary CD30+), two follicular lymphomas]. ALK− lymphomas with anaplastic morphology showed extra nonrearranged anaplastic lymphoma kinase gene loci (P=0.004) due to trisomy 2 irrespective of the following factors: B or T/null phenotype (P=0.315), diagnostic categories of systemic or cutaneous ALCL or the above-mentioned B-cell lymphomas (P=0.131), and CD30 positivity by immunohistochemistry (P=1.000). Trisomy 2 was absent in all ALK+ lymphomas (P=0.009), which showed rearranged ALK gene loci (P<0.001). Whether trisomy 2 is a primary or secondary event that leads to ALK− lymphomas cannot be determined from this study. Its presence in secondary B-cell lymphomas suggests that trisomy 2 may be a secondary cytogenetic aberration in lymphomas in general. Further investigation of this finding is necessary to further our understanding of the heterogeneous group of ALK− lymphomas.