Treatment of SCID/human B cell precursor ALL with anti-CD19 and anti-CD22 immunotoxins.

The anti-CD19 (HD37-dgRTA) and anti-CD22 (RFB4-dgRTA) immunotoxins (ITs) are murine IgG(1) monoclonal antibodies (Mabs) conjugated to a deglycosylated ricin A chain (dgRTA). They are effective in killing B-lineage non-Hodgkin's lymphoma (NHL) cells in vitro, in vivo and in adult patients with B-lineage NHL. The potential of these agents for the treatment of childhood B-precursor acute lymphoblastic leukemia (ALL) is unknown. The anti-CD19 and anti-CD22 ITs should have anti-tumor activity against childhood B-lineage ALL since both target antigens are expressed on the surface of these cells. We have previously shown that, in vitro these two ITs selectively kill leukemia cells obtained from children with leukemia. To evaluate the efficacy of our ITs in an in vivo model we injected the human pre-B ALL cell line, NALM-6-UM1, into severe combined immunodeficient (SCID) mice. We tested the ability of two ITs to prolong survival or cure mice of both early and advanced tumors. In early disease, treatment with HD37-dgRTA, RFB4-dgRTA, or Combotox (an equimolar concentration of the two ITs) significantly improved their survival. In advanced disease, treatment with RFB4-dgRTA or Combotox significantly improved survival. Overall there were 10 long-term survivors who were cured, as determined by survival beyond 150 days with no evidence of disease as determined by polymerase chain reaction (PCR) analysis.

Immunotoxins against CD19 and CD22 are effective in killing precursor-B acute lymphoblastic leukemia cells in vitro.

Monoclonal antibodies (Mabs) conjugated to toxins or their subunits (immunotoxins or ITs) are undergoing clinical testing in adults with a variety of malignancies. The potential impact of this form of therapy in pediatric precursor B-lineage acute lymphoblastic leukemia (pre-B ALL) has yet to be determined. Mabs directed against the cell surface antigens, CD19 and CD22 conjugated to deglycosylated ricin A chain (dgRTA) have been tested in patients with non-Hodgkin's lymphoma (NHL), but not in patients with pre-B ALL. Because of the encouraging performance of these ITs in phase I trials, we evaluated the specific cytotoxicity of anti-CD19 (HD37-dgRTA) and anti-CD22 (RFB4-dgRTA) ITs or their combination (Combotox) on patient-derived pre-B ALL cells maintained in vitro on a stromal feeder layer. After 48 h in culture, cytotoxicity to tumor cells was determined by flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated anti-CD10, 19, and 22. Both RFB4-dgRTA and HD37-dgRTA induced a statistically significant reduction in the number of viable leukemic cells, and Combotox was even more effective. Our results demonstrate that these ITs are specifically cytotoxic to primary pre-B ALL cells and that they should be further evaluated for the therapy of B-lineage ALL.

Transient myeloproliferative disorder and acute myeloid leukemia in Down syndrome. An immunophenotypic analysis.

Immunophenotypic analysis of transient myeloproliferative disorder (TMD) and acute myeloid leukemia (AML) using multiparameter flow cytometry might provide insight into their relationship. We retrospectively analyzed the expression of multiple lymphoid, myelomonocytic, and megakaryocytic antigens on blast proliferations in 18 patients with Down syndrome (DS; AML, 9; TMD, 9). The AMLs and TMDs shared several immunophenotypic characteristics. Blasts in all expressed CD45, CD38, and CD33; most AMLs and all TMDs were CD36+; and the majority expressed CD41 and CD61, suggesting megakaryocytic differentiation. The majority of cases were CD34+, CD14-, and CD64-. There was aberrant expression of the T-cell-associated antigen CD7 in most AMLs and TMDs. CD56 was expressed aberrantly in 5 AMLs and 7 TMDs. The major difference between the disorders was the pattern of expression of myeloid markers CD11b and CD13; each was expressed in 8 AMLs but only 2 TMDs. Blasts were HLA-DR-positive in 3 AMLs vs 7 TMDs. Blasts in TMD and AML in DS have a characteristic immunophenotype distinct from AML in other settings. The immunophenotypic similarities suggest a biologic relationship between the disorders; however, distinct immunophenotypic differences also were observed.

Immunophenotypic analysis of peripheral T-cell neoplasms. A multiparameter flow cytometric approach.

We retrospectively reviewed multiparameter flow cytometric analyses in 50 peripheral T-cell neoplasms (PTCNs). Results were interpreted within the context of a large cohort of nonneoplastic T-cell populations. All PTCN diagnoses were confirmed with morphologic and/or molecular analysis. Aberrant populations were defined as discrete immunophenotypic clusters exhibiting loss of or increased or diminished expression of T-cell antigens relative to internal immunophenotypically normal T-cell populations. An antigenic pattern was considered abnormal if it exceeded ranges for T-cell subsets in specific anatomic sites or was not normally encountered. Forty-six of 50 and 41 of 50 demonstrated 1 or more and 2 or more aberrations, respectively. The most common abnormally expressed antigen was CD3, followed by CD7, CD5, and CD2. Except for CD7, abnormally dim or bright antigen expression was more common than deletion. Only 3 cases were abnormal solely based on expansion of an otherwise immunophenotypically normal population; the remainder had patterns of antigen expression not seen in nonneoplastic populations. These data indicate that most PTCNs are aberrant by multiparameter flow analysis. However, results must be interpreted within the context of thorough knowledge of the immunophenotypic spectrum of nonneoplastic T cells.

De novo CD5+ diffuse large B-cell lymphomas. A heterogeneous group containing an unusual form of splenic lymphoma.

We reviewed our institutional experience with de novo CD5+, large B-cell lymphomas to determine whether they represent a distinct entity and are related to CD5+ small B-cell disorders. We identified 13 cases with multiparameter flow cytometry over a period of 58 months (5% of large B-cell lymphomas) in 7 females and 6 males. Three groups were identified. Group 1 (2 cases) had diffuse splenic red pulp involvement with a distinctive cordal pattern of infiltration, no other clinical evidence of mass disease, microscopic disseminated disease on further workup, and an identical immunoglobulin-negative immunophenotype. Group 2 cases (7 cases) were clinically and morphologically heterogeneous and had an immunophenotype resembling mantle cell lymphoma (FMC7-positive, CD23-). Group 3 (4 cases) had miscellaneous immunophenotypes, including one closely resembling chronic lymphocytic leukemia. Cyclin D1 was positive in only 1 of 10 evaluable cases (group 2). We conclude that CD5+ diffuse large B-cell lymphomas are heterogeneous; most cases do not seem to be related to chronic lymphocytic leukemia or mantle cell lymphoma. However, we identified a subgroup of primary splenic CD5+ large B-cell lymphoma with diffuse red pulp involvement and believe this may represent a distinct clinicopathologic entity.

Craniopagus parasiticus: a case illustrating its relationship to craniopagus conjoined twinning.

A case of craniopagus parasiticus is described in which the parasitic twin is more fully developed anatomically than in any of the previous reports. Somatic and placental vascular anastomoses between the twins and hypoplasia of the umbilical cord of the parasite were also observed. These findings support the hypothesis that craniopagus parasiticus results from compromise of the blood supply to one of a pair of craniopagus conjoined twins.

Prognostic significance of tumour-infiltrating T lymphocytes and T-cell subsets in de novo diffuse large B-cell lymphoma: a multiparameter flow cytometry study.

Tumour-infiltrating T lymphocytes (TIL-T) have been implicated in playing a role in controlling tumour growth. We evaluated TIL-T in 55 cases of de novo diffuse large B-cell lymphoma (DLBCL) using three- or four-colour flow cytometric immunophenotyping (FCI). The percentage of TIL-T varied from 3% to 72% of total viable cellular events (mean 32 +/- 20%). The CD4:CD8 ratio varied from 0.17 to 13 (mean 2.3 +/- 2.2). Cases with >/= 20% T cells and those with CD4:CD8 ratios > or = 2.0 showed a significantly better overall survival (P = 0.017 and P = 0.034 respectively). These findings were independent of clinical stage at diagnosis. The T-cell percentage and CD4:CD8 ratio were moderately correlated (Spearman correlation coefficient = 0.47, P = 0.001) and multivariate analysis revealed that the association of the two factors with prognosis was mutually dependent. The T cells in 23 cases were studied for CD45RO. The mean percentage of total T cells expressing CD45RO was 86 +/- 10%. There was a trend towards better survival for those patients with a higher percentage of CD45RO+ T cells (P = 0.06). These results suggest that TIL-T, particularly CD4+ T cells, may play a role in the control of DLBCL, and measurement of T-cell percentage and T-cell subsets using FCI may be useful in predicting the clinical behaviour of DLBCL.

Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4-color flow cytometry.

Bone marrow hematogones (B-lymphocyte precursors) may cause problems in diagnosis because of their morphologic and immunophenotypic similarities to neoplastic lymphoblasts. The purposes of this prospective, multiparametric flow cytometry study were to quantify hematogones across age groups and a spectrum of clinical conditions, to identify factors that affect the relative quantity of hematogones, and to compare their immunophenotype with that of neoplastic lymphoblasts. A total of 662 consecutive marrow specimens were analyzed for hematogones using one of two 4-color antibody combinations; hematogones were identified in 528 (79.8%). There was a significant decline in hematogones with increasing age (P <.001), but a broad range was found at all ages and many adults had a relatively high number. Specimens processed by density gradient had a higher mean percent hematogones than those processed by erythrocyte lysis (P <.001). There was a direct decline in hematogones with increasing marrow involvement with neoplastic cells. A total of 8% of the 662 specimens contained 5% or more hematogones: 24.6% of specimens from patients aged less than 16 years and 6.3% from those 16 and older (P <.000 01). Increased hematogones were observed most often in patients with lymphoma, marrow regenerative states, immune cytopenias, and acquired immunodeficiency syndrome. Hematogones always exhibited a typical complex spectrum of antigen expression that defines the normal antigenic evolution of B-cell precursors and lacked aberrant expression. In contrast, lymphoblasts in 49 cases of precursor B-ALL showed maturation arrest and exhibited 1 to 11 immunophenotypic aberrancies. Four-color flow cytometry with optimal combinations of antibodies consistently distinguishes between hematogones and neoplastic lymphoblasts.