A phase I-II study of rituximab, ifosfamide, mitoxantrone and etoposide (R-IME) for B cell non-Hodgkin's lymphoma prior to and after high-dose chemotherapy and autologous stem cell transplantation (HDC-ASCT).

This phase I-II study describes the safety of rituximab, ifosfamide, mitoxantrone and etoposide (R-IME) as an induction regimen prior to high-dose chemotherapy and autologous stem cell transplantation (HDC-ASCT), and rituximab given post-HDC-ASCT for B cell non-Hodgkins's lymphoma. This study also measured the effect on disease burden and stem cell contamination. Patients with relapsed, refractory or poor risk B cell lymphomas were eligible. Patients were treated with two cycles of R-IME; all non-progressing patients under-went a third cycle and peripheral blood stem cell (PBSC) collection. Patients underwent HDC-ASCT and those patients in remission after HDC-ASCT were treated with four additional doses of rituximab. Tumor cell contamination was measured at baseline and in the PBSC. Serial immunoglobulin levels were measured. Patients were followed for time to treatment failure (TTF) and overall survival (OS). Thirty-two patients were enrolled. Thirty patients had at least stable disease after two cycles of R-IME. Twenty-nine underwent stem cell collection. The response rate to R-IME induction was 77% (20/26) with 35% (9/26) complete response(CR). Stem cell mobilization was successful in 93% (27/29) of patients. The response rate to R-IME induction and HDC-ASCT was 95% with a confirmed CR of 68%. Median follow-up was 28 months; the median TTFand OS have not been reached. There was a significant decline in stem cell tumor cell contamination and a significant decline in IgG without an increase in infections. Forty-three per cent of patients had transient neutropenia after post-transplant rituximab. R-IME is an effective cytoreductive and mobilization regimen. There appears to be a reduction in the number of lymphoma cells in the stem cell product and the toxicity is manageable.

Rituximab and ifosfamide, mitoxantrone, etoposide (RIME) with Neupogen support for B-cell non-Hodgkin's lymphoma prior to high-dose chemotherapy with autologous haematopoietic transplant.

A phase I/II study was performed to analyse the ability of ifosfamide-based chemotherapy with rituximab to produce a turmour-free graft as well as the safety of retuximab prior to stem cell harvest and post high-dose chemotherapy. Twenty-two patients with B-cell non-Hodgkin's lymphoma were enrolled either having aggressive large-cell disease in relapse or at high/high-intermediate risk of relapse, or refractory lymphoma or mantle cell lymphoma, or indolent lymphoma. Chemotherapy consisted of ifosfamide 2 g/m2, days 1-3 with mesna, etoposide 100 mg/m2, days 1-3, and mitoxantrone 8 mg/m2 day 1, with figrastim. Rituximab was given at 375 mg/m2 for 4 doses. An encouraging overall response rate of 90%, including 11 CRs was achieved. CD34+ cells were successfully mobilized in 18 or 19 patients analysed so far with a median number of 3.4 x 10(6) cells/kg. The combination of ifosfamide-based chemotherapy with rituximab significantly reduced the number of contaminating B-cells in the stem cell product and so far there has only been a single relapse post high-dose chemotherapy with autologous haematopoietic transplant. The RIME regimen was generally well tolerated with minimal non-haematological toxicity and most of the treatment was done completely on an outpatient basis. Haematological toxicity was manageable with filgrastim, there were some infectious complications.

Breast tumor contamination of PBSC harvests: tumor depletion by positive selection of CD34(+) cells.

BACKGROUND: Positive selection of CD34(+) cells may reduce or eliminate tumor cells contaminating PBSC harvests of breast cancer (BrCa) patients. However, to assess tumor purging accurately methods may be needed that are of higher sensitivity than standard immunocytochemistry (ICC) assays. METHODS: BrCa-cell depletion, resulting from CD34(+) cell selection, was evaluated using a novel, highly sensitive assay based upon immunomagnetic enrichment with ICC detection in 36 BrCa patients undergoing highdose chemotherapy with autologous PBSC support. RESULTS: The prevalence of BrCa-cell contamination was significantly lower (P = 0.0078) in selected CD34(+) cell fractions (17/35, 49%) from apheresis collections compared with CD34(-) cell fractions (25/35, 71%). In 8/34 (24%) patients, BrCa cells were detected in CD34(-) cell fractions, but not in paired CD34(+) cell fractions. Significantly lower total numbers (P < 0.0005) of BrCa cells were enumerable in CD34(+) cell fractions compared with corresponding apheresis harvests. The median total BrCa-cell content of selected CD34(+) cell fractions with measurable contamination was 22 BrCa cells (range, 6-73 BrCa cells), compared with 3297 BrCa cells (range, 10-98 400 BrCa cells) in apheresis harvests. The median log depletion of BrCa cells achieved by positive CD34(+) cell selection in specimens with detectable contamination both before and after selection was 2.2 (range, 1.7-4.0). Total pre-selection BrCa cell number was significantly predictive (P = 0.004) of residual detectable post-selection contamination. DISCUSSION: Positive CD34(+) cell selection is an effective tumor purging strategy. The prevalence of PBSC contamination in BrCa patients is substantially higher than formerly appreciated.

Breast tumor contamination of peripheral blood stem cell harvests: increased sensitivity of detection using immunomagnetic enrichment.

Contaminating tumor cells in peripheral blood stem cell (PBSC) grafts infused for hematopoietic rescue after high-dose chemotherapy could potentially contribute to relapse in BrCa patients. To date the prevalence of PBSC contamination in BrCa patients, as determined by standard immuno-cytochemistry (ICC) assays, has generally been found to be relatively low. However, assay sensitivity may have an important impact on the ability to detect contamination. In this investigation a novel and highly sensitive BrCa cell assay using immunomagnetic enrichment with a panel of antiBrCa monoclonal antibodies (MAbs) and detection by ICC has been characterized. The immunomagnetic enrichment with ICC detection (IE-ICC) assay was directly compared with standard ICC in ability to detect BrCa contamination of PBSC specimens from patients with high-risk stage II/III and metastatic disease. The sensitivity of the IE-ICC assay was approximately 50-fold greater than that of standard ICC. As determined by standard ICC assay, BrCa cells were present in 1/14 patients (7%) and 2/26 (8%) specimens. In contrast, with IE-ICC assay the proportions of positive findings in patients (12/14, 86%) and specimens (19/26, 73%) were significantly higher (p = 0.001 and p < 0.0005, respectively). These preliminary findings suggest that the prevalence of PBSC contamination may be substantially higher than previously appreciated. Consequently, measures to reduce tumor contamination in the graft may have the potential to improve patient outcomes. Higher sensitivity assays such as the IE-ICC assay may play an important role in assessing the risks associated with tumor contamination and the effectiveness of tumor-purging approaches such as positive selection of CD34+ cells and in monitoring patient response to therapy.

Alterations of differentiation, clonal proliferation, cell cycle progression and bcl-2 expression in RAR alpha-altered sublines of HL-60.

All-trans retinoic acid (RA) induces granulocytic differentiation of acute promyelocytic leukemia cells both in vivo and in vitro. In the HL-60 wild-type (WT) early promyelocytic leukemia cell line, granulocytic differentiation appears to be directly mediated by the nuclear receptor RAR alpha. An HL-60 subline resistant to RA (HL-60 R) contains a point mutation which results in a truncation of 52 amino acids at the COOH end of RAR alpha. Cross-talk between differentiation, clonal inhibition of growth and apoptosis was studied using HL-60 WT, HL-60 R, and HL-60 R infected by a retroviral vector containing RAR alpha (LX) as targets, which were cultured with various retinoids, vitamin D3 analogs, HMBA, or DMSO. None of these compounds induced significant differentiation of HL-60 R and HL-60 LX, but they did induce differentiation of HL-60 WT. In contrast, retinoids inhibited the clonal proliferation of HL-60 WT, HL-60 R, and HL-60 LX. Vitamin D3 analogs including KH1060 stimulated the clonal growth of HL-60 R; but they inhibited clonal growth of HL-60 WT and LX. Levels of Bcl-2 strongly decreased in HL-60 WT and LX after treatment by retinoids, while no change in expression occurred in HL-60 R. Neither KH 1060 nor 9-cis RA induced apoptosis of HL-60 R, but these agents did induce apoptosis in HL-60 LX WT. Taken together, we showed that HL-60 R has a global defect in its ability to be induced to differentiate by a variety of pathways, not merely the retinoid pathway. Furthermore, our HL-60 models showed that inhibition of proliferation and induction of apoptosis and differentiation can be dissociated. Clinically, these results suggest that several putative differentiation agents may have anti-cancer (antiproliferative) activities, even though they do not induce differentiation of the cancer cells.

Synergistic decrease of clonal proliferation, induction of differentiation, and apoptosis of acute promyelocytic leukemia cells after combined treatment with novel 20-epi vitamin D3 analogs and 9-cis retinoic acid.

Patients with acute promyelocytic leukemia (APL) usually relapse after all-trans retinoic acid (RA) treatment because this therapy fails to eradicate the malignant clone. Our data showed that KH 1060 and other 20-epi vitamin D3 analogs alone were potent inhibitors of clonal growth of NB4 cells, an APL cell line (ED50, approximately 5 x 10(-11) M). The combination of KH 1060 and 9-cis-RA synergistically and irreversibly enhanced this effect. Neither KH 1060 nor 9-cis-RA (10(-6) M, 3 d) were strong inducers of differentiation of NB4 cells. However, 98% of the cells underwent differentiation to a mature phenotype with features of both granulocytes and monocytes after exposure to a combination of both compounds. Apoptosis only increased after incubation of NB4 cells with 9-cis-RA alone (28%) or with a combination of 9-cis-RA plus KH1060 (32%). Immunohistochemistry showed that the bcl-2 protein decreased from nearly 100% of the wild-type NB4 cells to 2% after incubation with a combination of KH 1060 and 9-cis-RA, and the bax protein increased from 50% of wild-type NB4 cells to 92% after culture with both analogs (5 x 10(-7) M, 3 d). Western blot analysis paralleled these results. Studies of APL cells from one untreated individual paralleled our results with NB4 cells. Taken together, the data demonstrated that nearly all of the NB4 cells can be irreversibly induced to differentiate terminally when exposed to the combination of KH 1060 and 9-cis-RA.

Potent tumoricidal effects of a human cytotoxic T-cell line (TALL-104) against prostate cancer.

The human TALL-104 cell line possesses major histocompatibility complex non-restricted cytotoxic activity against a large variety of tumor targets. Adequate therapies for prostate cancer that has spread outside its capsule are lacking. In order to identify effective therapies for this problem, we investigated the antiproliferative effects of TALL-104 cells against three prostate cancer cell lines (LNCaP, PC-3, DU-145). A Cr-51-release: cytotoxicity assay showed that TALL-104 cells were very cytotoxic against the prostate cancer cells. For example, at a 1:1 ratio of TALL-104 cells to prostate cancer cells, the percent release of Cr-51 at 18 h were 50, 40, and 45% for LNCaP, PC-3, and DU-145, respectively. Analysis by inhibition of clonogenic growth of prostate cancer cells also showed that TALL-104 cells were extremely effective. For instance, a short-term (4 h or 18 h) pre-incubation of TALL-104 cells with these tumor cells at the effector to target ratio of 10:1 prior to clonogenic assay resulted in a substantial reduction in clonogenic tumor growth (90%, 65%, and 50% clonal growth inhibition for LNCaP, PC-3, and DU-145, respectively). Further experiments using both Cr-51 release and clonogenic assays showed that irradiated TALL-104 cells were also effective in their anti-prostatic cancer activities. We also examined if TALL-104 cells plus a chemotherapeutic agent might complement each other in their cytotoxic effects. Preincubation of prostate cancer cell targets with etoposide (0.2-20 mu g/ml) for 18 h markedly increased their susceptibility to TALL-104 lysis. The anti-tumor efficacy of TALL-104 cells was also demonstrated in vivo utilizing the BNX murine model engrafted with subcutaneous PC-3 prostate cancer cells. A substantial reduction in PC-3 tumor cell progression was observed in mice injected with irradiated TALL-104 cells (1x10(7) cells intraperitoneally or intratumorally for 5 days beginning on days 24 and 45 after implantation) as compared to mice injected with tumors only. Taken together, these findings suggest that TALL-104 cells may be utilized as a potent anti-tumor agent, either alone or in combination with other agents (such as etoposide) in metastatic prostate cancer.

Combination of a potent 20-epi-vitamin D3 analogue (KH 1060) with 9-cis-retinoic acid irreversibly inhibits clonal growth, decreases bcl-2 expression, and induces apoptosis in HL-60 leukemic cells.

All-trans retinoic acid (RA) is the first highly effective differentiation-inducing agent for remission induction in patients with acute promyelocytic leukemia. However, remissions are short-lived because the treatment fails to induce complete differentiation and fails to eradicate the malignant clone. To eliminate rapidly the malignant clone, in analogy with aggressive chemotherapy, the combination of potent differentiation- and apoptosis-inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 (1,25-dihydroxyvitamin D3; 1,25(OH)2D3) inhibits proliferation and induces differentiation of myeloid leukemic cells. The 9-cis-RA, unlike all-trans-RA which binds only retinoic acid receptors, is a high affinity ligand for both retinoic acid receptors and retinoid X receptors. The aim of this study was to evaluate the therapeutic potential of combining a vitamin D(3) analogue, 20-epi-22-oxa-24a,26a,27a-tri-homo-1alpha,25(OH) 2D, (KH 1060), which belongs to the family of potent 20-epi-1,25(OH),D3 analogues, with 9-cis-RA by assessing their effects on the proliferation, differentiation, and apoptosis of the human leukemia cell line HL-60 in vitro. Our data show that KH 1060 alone is a very potent inhibitor of clonal proliferation of HL-60, but this effect is reversible, and that 9-cis-RA alone is a weak inhibitor of clonal proliferation of HL-60 cells. In contrast, the combination of KH 1060 and 9-cis-RA synergistically and irreversibly inhibited the clonal proliferation of HL-60 cells and induced apoptosis, as detected by morphological changes and DNA fragmentation. This combination also affected the expression of apoptosis-related genes. The bcl-2 protein became nearly undetectable, and expression of bax protein increased slightly (the bax:bcl-2 ratio was 14-fold higher than in untreated cells). Differentiation of treated HL-60 cells was assessed by their ability to produce superoxide, as measured by reduction of nitro blue tetrazolium, positive staining for alpha-naphthyl acetate esterase, phagocytosis, morphology, and analysis of membrane-bound differentiation markers with two-color immunofluorescence. Treatment with the combination of KH 1060 and 9-cis-RA was a potent inducer of differentiation of HL-60, with the cells developing a myelomonocytic phenotype. In summary, our data demonstrate that the combination of both KH 1060 and 9-cis-RA irreversibly and synergistically inhibited clonal growth, induced differentiation and apoptosis of HL-60 cells concomitantly with a very marked decreased expression of bcl-2, and increased the bax:bcl-2 ratio. This drug combination may have important therapeutic significance.

20-epi-vitamin D3 analogues: a novel class of potent inhibitors of proliferation and inducers of differentiation of human breast cancer cell lines.

We have studied the in vitro biological activities and mechanism of action of 1,25-dihydroxyvitamin D3 (1,25D3) and four potent 1,25D3 analogues [20-epi-22oxa-24a,26a,27a-tri-homo-1,25(OH)2D3 (KH 1060); 20-epi-1,25(OH)2D3; 1,25(OH)2-16ene-D3; and 1,25(OH)2-16ene-23yne-D3] on proliferation and differentiation of estrogen receptor-negative (MDA-MB-436, BT-20, SK-BR-3, and MDA-MB-231), estrogen receptor-weakly positive (BT474), and estrogen receptor-positive (MCF-7) breast cancer cell lines. Dose-response studies showed that KH 1060 was the most potent analogue, because it was able to induce differentiation in all seven breast cancer cell lines (measured by lipid staining) and to suppress more than 50% clonal proliferation (ED50) at 10(-10) M in all cell lines, except MDA-MB-436 and BT-20. To explore how these compounds mediated antiproliferative actions, their effects on the cell cycle, on expression of bcl-2 and p53, and on apoptosis were assessed. Five of six cell lines have a mutant p53 gene, whereas MCF-7 has wild-type p53. Immunohistochemical staining showed that the p53 protein was predominantly localized in the nucleus in each of the breast cancer cell lines except for MCF-7, which expressed the protein predominantly in the cytoplasm. After incubation with KH 1060 (3 days; 10(-7) M), expression of bcl-2 protein as determined by immunohistochemical localization was markedly decreased in BT-474, MCF-7, and MDA-MB-231; these same cells were profoundly inhibited in their clonal proliferation and arrested in the G0/G1 phase of the cell cycle when cultured with KH 1060. In contrast, BT-20 and MDA-MB-436 cells that were refractory to the antiproliferative effect of KH 1060 (ED50 < 10(-6) M) had no down-regulation of their bcl-2 expression and no cell cycle changes after exposure to KH 1060. MCF-7 showed morphological changes and DNA fragmentation, indicative of apoptosis after 48 h incubation with KH 1060 (10(-6) M), during which time p53 protein accumulated in the nucleus and decreased in the cytoplasm. In contrast, no apoptosis was detected in three other breast lines (MDA-MB-231, SK-BR-3, and BT-474) that had a mutated p53. In conclusion, the data indicate that KH 1060 is an extremely potent 1,25D3 analogue inducing differentiation of all six breast cancer lines and potently inhibiting clonal growth of four of them with concomitant decreased bcl-2 and cell cycle arrest at G0/G1.(ABSTRACT TRUNCATED AT 400 WORDS)

Transient erythroblastopenia of childhood. Evidence for cell-mediated suppression of erythropoiesis.

PURPOSE: T cell-mediated red cell aplasia in a 4 1/2-year-old child with transient erythroblastopenia of childhood (TEC) is described. PATIENTS AND METHODS: Erythropoiesis was studied by assessing the colony growth of marrow erythroid progenitors at the time of diagnosis and during recovery. RESULTS: The colony-forming unit-erythroid (CFU-E) growth of whole marrow at diagnosis was only 28% that of the control. T-cell depletion of the patient's marrow was followed by a more than fivefold increase in CFU-E growth, as compared with 20% inhibition of CFU-E and 40% inhibition of burst-forming unit-erythroid (BFU-E) growth in control marrow. The number of colony-forming unit-granulocyte-macrophage (CFU-GM) in both control and patient's marrow was not significantly altered by all of these manipulations. During early and late recovery, CFU-E and BFU-E growth improved substantially, and the effect of T-cell depletion diminished. Increased numbers of peripheral T-suppressor lymphocytes, as well as activation of natural killer (NK) cells and high levels of interferon, all consistent with viral infection, were found at presentation. Clinical recovery was associated with normalization of T-suppressor lymphocyte number. CONCLUSIONS: The results suggest that in this child with TEC, a preceding viral infection may have caused activation of suppressor T-cells and interferon secretion leading to cell-mediated suppression of erythropoiesis.

Establishment and characterization of a human mixed-lineage, T-lymphoid/myeloid cell line (USP-91).

We report the establishment of a novel cell line from a pediatric patient with recurrent non-Hodgkin's lymphoma. This cell line, termed USP-91, showed both T-lymphoid cell as well as myeloid (ie, nonlymphoid) cell characteristics using a comprehensive multiparameter approach. The initial growth of this cell line was dependent on the presence of the murine stromal cell line, 14F1.1. Subsequently, a phenotypically stable, stroma-independent cell line was established. Although the recurrent biopsy material and the derivative cell line, USP-91, were clonally-derived from T-lineage lymphoid cells, as evidenced by the same rearrangement of the T-cell receptor-beta locus, USP-91 coexpressed both the T-cell antigens CD7, CD3, and CD4, and the myeloid antigens CD13, CD33, CD11b, and CD34. The myeloid features of USP-91 were most consistent with monocytic differentiation as these cells expressed alpha-napthol acetate esterase, lysozyme, alpha-1-antitrypsin, alpha-1-antichymotrypsin, as well as the cell surface receptor for macrophage colony-stimulating factor. In addition, incubation in the presence of phorbol esters induced USP-91 to exhibit morphologic and functional properties of mature mononuclear phagocytes. The expression of this bilineage phenotype suggests that USP-91 represents the malignant transformation of a progenitor cell capable of either myelomonocytic or T-lymphoid differentiation.

Recombination activating gene-1 (RAG-1) expression in all differentiation stages of B-lineage precursor acute lymphoblastic leukemia.

The recombination activating gene-1 (RAG-1), which is required for immunoglobulin (Ig) gene rearrangement, is expressed in murine B-lymphoid precursors but not in mature B lymphocytes. In order to characterize the temporal relationship of RAG-1 expression to other markers of human B-lymphoid differentiation [cell surface antigens, terminal deoxynucleotidyl transferase (TdT), Ig gene rearrangements], RAG-1 expression was studied in a group of B lineage childhood acute lymphoblastic leukemia (ALL). ALL cells from 21 patients were grouped into three developmentally related phenotypes based on the expression of the differentiation antigens CD19, CD10, and CD20. All 21 leukemias were surface Ig (slg) negative. There were leukemias representing each developmental stage of Ig gene rearrangement. RAG-1 was expressed in 20 of 21 B-lineage ALL, including leukemic cells from each stage of differentiation, as defined by immunophenotype and IgH and IgL gene rearrangement status. RAG-1 was expressed in slg- ALL, regardless of the Ig heavy chain (IgH) or Ig light chain (IgL) gene configuration. RAG-1 was not expressed in two Burkitt lymphomas and Burkitt lymphoma cell lines with slg+ mature B-lymphocyte phenotype. In two cases, RAG-1 was expressed in TdT-negative ALL; conversely TdT was expressed in the one RAG-1 negative ALL. These results suggest that RAG-1 in B-lineage ALL is expressed at all phenotypic and genotypic developmental stages preceding surface immunoglobulin expression, and that TdT and RAG-1 may be regulated by different mechanisms.

Aminopeptidase P and dipeptidyl peptidase IV activity in human leukocytes and in stimulated lymphocytes.

Human white blood cells were shown to contain high aminopeptidase P activity. The specific activities found in the high-speed supernatant of the extracts of granulocytes, lymphocytes and monocytes ranged from 30 to 70 units per mg protein. Culturing lymphocytes during 7 days in the presence of phytohaemagglutinin resulted in a 70-200% increase in the specific aminopeptidase P activity and a 200% increase in the specific activity of dipeptidyl peptidase IV. The time-course of the activity of both aminopeptidase P and dipeptidyl peptidase IV during the stimulation of human T-lymphocytes by phytohaemagglutinin indicates an involvement of these two enzymes in the proliferative process of these immunocompetent cells. Due to their substrate specificity their potential substrates must have the N-terminal Xaa-Pro sequence known to be present in several immunologically important polypeptides.

Recombinant interleukin-2-activated intracavitary lymphocytes: phenotypic characteristics and effector function.

A preclinical study of intracavitary lymphocytes (ICL) from malignant effusions of cancer patients is described. The object of this study was to evaluate the antitumor potential of ICL as a baseline for developing adoptive immunotherapy trial for ovarian carcinoma patients. The main parameters studied were functional cytolytic activity of fresh and recombinant interleukin-2 (rIL-2)-activated ICL and their phenotypic characteristics. Spontaneous cytolytic activity of ICL was detected in all samples tested against natural killer (NK)-sensitive targets (K562), while very low activity was shown against NK-resistant targets (Daudi) and fresh tumor cells. Activation in culture with rIL-2 generated cytolytic activity against NK-resistant targets and significantly augmented NK activity. The pattern of antitumor lytic activity of ICL resembles lymphokine-activated killer cell activity and is non-major histocompatibility complex restricted against a variety of tumor targets. Phenotypic characterization of fresh ICL showed the predominance of CD3+ cells with the CD4/CD8 ratio resembling that of peripheral blood lymphocytes. During culture with rIL-2, changes in phenotypic expression of activated ICL were detected: enrichment in NKH1+ cells (up to 65%), of CD8+ (up to 70%), and very late antigen (VLA)-1+ cells (up to 54%), concomitantly with a decrease in CD4+ population. The NKH1, CD8, and VLA-1 expression peaked at 2 weeks in culture and coincided with peak cytolytic activity of cultured ICL. Depletion of CD8+ cells from activated ICL resulted in a decreased proportion of cells expressing the NKH1 and VLA-1 phenotype, whereas depletion CD4+ cells led to enrichment in CD8, NKH1, and VLA-1 antigens. Cytolytic activity was significantly increased in CD4 depleted population against NK-resistant and NK-sensitive targets. In this study we found that rIL-2 activation and culture of ICL generates killer activity against NK-resistant and fresh tumor targets and that enrichment in expression of NKH1, CD8, and VLA-1 antigens is associated with effector function.

Biologic and cytogenetic characteristics of leukemia in infants.

Clinical features, leukemic cell characterization, chromosomal findings, and treatment outcome were analyzed in a retrospective study of 30 cases with acute leukemia of infancy, 24 infants with acute lymphoblastic leukemia (ALL), and six cases with acute nonlymphoblastic leukemia (ANLL). Extensive bulky disease with organomegaly, central nervous system (CNS), and skin involvement were prominent features at diagnosis with a higher frequency in ANLL as compared to ALL. Four of six ANLL patients were classified as monocytic or myelomonocytic. In the ALL group nine of 24 (36%) were non-L1 morphology and six of 17 (33%) were common ALL antigen (CALLA) negative, the majority of them (five of six) were included in the non-L1 group. Immunophenotyping revealed four cases with early B-cell (three patients: Ia+B4+, and one patient: Ia+) and two cases with T-cell. Mixed lineage leukemia was found in five infants. Heavy chain immunoglobulin gene rearrangement was present in six cases tested, two CALLA+, two with Ia+B4+, and two were undifferentiated mixed lineage leukemia. Chromosomal aberrations were detected in ten of 18 patients, mostly in ANLL and CALLA negative ALL. Translocations were detected in six patients, involving 4q21-23 and 11q23 in three and two cases, respectively. The probability of five-year DFS were 27% for the whole group. The worst prognosis was observed in infants younger than 6 months of age, in whom the leukemia cell characteristics was compatible with stem cell: ANLL, very early pre-B, or undifferentiated mixed type. The chromosomal aberrations found in all cases included translocation with the seemingly nonrandom breakpoints at 4q21 and 11q23, and breakpoints that corresponded to known fragile sites. This finding may be suggestive of an underlying genetic predisposition associated with the poor prognosis of leukemia of infancy.

Heterogeneity of childhood acute lymphoblastic leukemia (ALL): monoclonal antibodies phenotyping and induction of differentiation in vitro.

Fifty-eight pediatric patients with non-T acute lymphoblastic leukemia (ALL) were diagnosed and evaluated at the Sambur Center of Pediatric Hematology Oncology. At least six subtypes of non-T ALLs were identified, corresponding to the various stages of B-cell differentiation, by utilizing an extensive panel of monoclonal antibodies directed against T- B- and myeloid-cell differentiation antigens. Moreover, leukemic cells expressing the phenotype of early B cells could be driven to differentiate along the B- cell lineage to express CALLA and BL antigens and cytoplasmic and/or surface immunoglobulins (IgM). A unique phenotype of non-T ALL was also identified. These leukemic cells expressed B cell antigen exclusively, i.e., HLA/DR and B4 (CD19). Myeloid-cell antigens, however, were expressed on these cells spontaneously after a 24-hour incubation in culture medium in vitro. In addition, leukemic cells of four patients with a phenotype of HLA/DR, CD19, and CD10 expressed antigens of the T-cell lineage: CD7 (3AI) and CD2 (leu 5), and/or of the myeloid cell lineage (My7). These results provide confirming evidence for the wide scope of the heterogeneity of ALL. It stresses the validity of accurate classification of leukemia to identify biologically and clinically unique subtypes of ALL, which bears specific prognostic parameters; and designates therapeutic protocols.

More than two immunoglobulin heavy chain J region genes in the majority of infant leukemia.

Congenital and infant leukemia are rare conditions associated with a very poor prognosis due to the high frequency of adverse clinical and laboratory parameters. As the occurrence of multiple immunoglobulin heavy chain hybridization band in childhood leukemia has been associated with poor prognosis, we studied whether it was present in this type of leukemia as well. Seven cases were examined, 4 of them less than 7 months of age. The immunophenotype was lymphoid in 5 and hybrid in 2. Most had abnormal karyotypes. In 5 of the 7, including all with congenital leukemia, an immunoglobulin heavy chain J region multiband pattern was found by Southern blot. The multiband pattern, whether primary or due to clonal evolution, seems to be associated with poor prognosis.

Immunologic dysregulation in a patient with familial hemophagocytic lymphohistiocytosis.

A 6-year-old Jewish Iranian girl with familial hemophagocytic lymphohistiocytosis (FHLH) is described. The course of the disease fluctuated with partial initial response to antibiotics, steroids, and supportive treatment. Subsequent cytotoxic treatment, including VP-16, Velban (vinblastine sulfate), and methotrexate (MTX) controlled the disease for a few months but the child died with a clinical picture of meningocephalitis 1.5 years later. Benign-looking lymphohistiocytic infiltrates with varying degrees of hemophagocytosis were present in the bone marrow, pleural effusion, cerebrospinal fluid (CSF), liver, and brain. Clinical and laboratory evidence of immunologic dysregulation during the disease could be demonstrated. Frequent and intense viral and bacterial infectious diseases were encountered. The laboratory examination most consistently found was the absence of natural killer (NK) cell activity against K562 target cells. The impaired activity of NK cells persisted during all stages of the disease including remission, although NK cell numbers, determined morphologically and immunophenotypically (by Leu-11, Leu-7), were normal. Natural killer activity could not be restored by interferon. Moreover, the interferon system appeared to be intact. Impaired monokin interleukin 1 (IL-I) production by peripheral blood monocytes was found and could not be restored by indomethacin. Lymphopenia, a mild decrease in T4 numbers, and subsequently, decreased proliferative response to mitogens was noted. Elevated immunoglobulin levels were found during exacerbations and viral episodes, at times accompanied by the presence of auto-antibodies. The exaggerated fatal lymphohistiocytic response typical for FHLH could be attributed to a underlying genetic pathologic dysregulation of the various immunological response pathways.

Undifferentiated leukemia of infancy with t(11:17) chromosomal rearrangement. Coexpressing myeloid and B cell restricted antigens.

It has been suggested that the malignant transformation, in some of the acute leukemias, may involve totipotent stem cells resulting in a biphenotypic leukemia expressing both myeloid, and lymphoid characteristics. We describe here a hybrid cell acute leukemia, in a 16-day-old infant, in whom leukemic cells coexpressed myeloid and lymphoid B cell antigens. Blast cells in the bone marrow showed L2 morphology according to the French American British (FAB) classification, with positive periodic-acid Schiff, and nonspecific esterase staining. Sudan black, and specific esterase were negative. Terminal deoxynucleotidyl transferase, was strongly positive in 5% of blasts, and faintly reactive with the rest. Karyotypic analysis demonstrated a translocation of t(11:17);(q23;p13). Immunoglobulin gene analysis revealed rearrangement of the heavy chain genes. The blasts' phenotype was HLA/DR+ B4+ My7+ My9+ common acute lymphoblastic leukemia antigen (CALLA) B1- T11-. Dual immunofluorescence staining using anti My7, and My9 fluorescein isothiocyanate, and anti B4 pycoerythrin conjugated monoclonal antibodies, and flow cytofluorometry, revealed a labeling pattern of 25% B4+; 10% to 15% My7+; 17% My9+; and 50% of cells coexpressing B4 My7, and My9 antigens. These results provide evidence for a hybrid leukemia with lymphomyeloblasts being part of a single clone, which may indicate the origin of this leukemic clone from a pluripotent (lymphoid/myeloid) stem cell.