Stromal interaction essential for vascular endothelial growth factor A-induced tumour growth via transforming growth factor-ß signalling.

BACKGROUND: High vascular endothelial growth factor (VEGFA) levels at the time of diagnosis confer a worse prognosis to multiple malignancies. Our aim was to investigate the role of VEGFA in promoting tumour growth through interaction with its environment. METHODS: HL-60 cells were transduced with VEGFA165 or control vector using retroviral constructs. Control cells (n=7) or VEGFA165 cells (n=7) were subcutaneously injected into NOD/SCID mice. Immunohistochemistry of markers for angiogenesis (CD31) and cell proliferation (Ki67) and gene expression profiling of tumours were performed. Paracrine effects were investigated by mouse-specific cytokine arrays. RESULTS: In vivo we observed a twofold increase in tumour weight when VEGFA165 was overexpressed (P=0.001), combined with increased angiogenesis (P=0.002) and enhanced tumour cell proliferation (P=0.001). Gene expression profiling revealed human genes involved in TGF-ß signalling differentially expressed between both tumour groups, that is, TGFBR2 and SMAD5 were lower expressed whereas the inhibitory SMAD7 was higher expressed with VEGFA165. An increased expression of mouse-derived cytokines IFNG and interleukin 7 was found in VEGFA165 tumours, both described to induce SMAD7 expression. CONCLUSION: These results suggest a role for VEGFA-driven tumour growth by TGF-ß signalling inhibition via paracrine mechanisms in vivo, and underscore the importance of stromal interaction in the VEGFA-induced phenotype.

Lactose maldigestion during methotrexate-induced gastrointestinal mucositis in a rat model.

Patients with chemotherapy-induced gastrointestinal mucositis suffer from anorexia, diarrhea, and stomach pain, often causing weight loss and malnutrition. When the intestinal function during mucositis would be known, a rational feeding strategy might improve the nutritional state, accelerate recuperation, and increase survival of mucositis patients. We developed a methotrexate (MTX)-induced mucositis rat model to study nutrient digestion and absorption. To determine lactose digestion and absorption of its derivative glucose during mucositis, we injected Wistar rats intravenously with MTX (60 mg/kg) or 0.9% NaCl (controls). Four days later, we orally administered trace amounts of [1-(13)C]lactose and [U-(13)C]glucose and quantified the appearance of labeled glucose in the blood for 3 h. Finally, we determined plasma citrulline level and harvested the small intestine to assess histology, myeloperoxidase level, glycohydrolase activity, immunohistochemical protein, and mRNA expression. MTX-treated rats showed profound villus atrophy and epithelial damage. During the experimental period, the absorption of lactose-derived [1-(13)C]glucose was 4.2-fold decreased in MTX-treated rats compared with controls (P < 0.01). Lactose-derived [1-(13)C]glucose absorption correlated strongly with villus length (rho = 0.86, P < 0.001) and with plasma citrulline level (rho = 0.81, P < 0.001). MTX treatment decreased jejunal lactase activity (19.5-fold, P < 0.01) and immunohistochemical protein and mRNA expression (39.7-fold, P < 0.01) compared with controls. Interestingly, MTX treatment did not affect the absorption of [U-(13)C]glucose during the experimental period. We conclude that lactose digestion is severely decreased during mucositis while glucose absorption is still intact, when supplied in trace amounts. Plasma citrulline level might be a useful objective, noninvasive marker for lactose maldigestion during mucositis in clinic.

Vascular endothelial growth factor receptor 2 (VEGFR-2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells.

AIMS: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. METHODS: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. RESULTS: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. CONCLUSIONS: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours.

Denaturing gradient gel electrophoresis of PCR-amplified gki genes: a new technique for tracking streptococci.

Viridans group streptococci (VGS) are a well-known cause of infections in immunocompromised patients, accounting for severe morbidity and mortality. Streptococcus mitis group species (Streptococcus mitis, Streptococcus pneumoniae, Streptococcus oralis) are among the VGS most often encountered in clinical practice. Identifying the portal of entry for S. mitis group strains is crucial for interventions preventing bacterial translocation. Unfortunately, tracking the source of S. mitis group strains is dependent on a combination of extremely laborious and time-consuming cultivation and molecular techniques (enterobacterial repetitive intergenic consensus-PCR [ERIC-PCR]). To simplify this procedure, a PCR analysis with newly designed primers targeting the household gene glucose kinase (gki) was used in combination with denaturing gradient gel electrophoresis (DGGE). This gki-PCR-DGGE technique proved to be specific for S. mitis group strains. Moreover, these strains could be detected in samples comprised of highly diverse microbiota, without prior cultivation. To study the feasibility of this new approach, a pilot study was performed. This confirmed that the source of S. mitis group bacteremia in pediatric patients with acute myeloid leukemia could be tracked back to the throat in five out of six episodes of bacteremia, despite the fact that throat samples are polymicrobial samples containing multiple S. mitis group strains. In contrast, using the classical combination of cultivation techniques and ERIC-PCR, we could detect these strains in only two out of six cases, showing the superiority of the newly developed technique. The new gki-PCR-DGGE technique can track the source of S. mitis group strains in polymicrobial samples without prior cultivation. Therefore, it is a valuable tool in future epidemiological studies.

Recurrent thrombo-embolism in a child with a congenital disorder of glycosylation (CDG) type Ib and treatment with mannose.

Thrombosis is a multifactorial disorder. Congenital disorders of glycosylation (CDG) are one of the known risk factors for its occurrence. These disorders result in glycosylation defects of glycoproteins, including those of the (anti-)coagulation system. CDG-Ib can specifically be treated with mannose, as illustrated by the case of a 4-year-old girl in whom deep venous thrombosis was the presenting symptom after a common viral infection. The diagnosis was made after recurrent episodes of thrombo-embolism and consumptive coagulopathy. After treatment with mannose no such episodes recurred. The pathophysiology of CDG as a risk factor for thrombotic disease is discussed.

Changes in body composition after childhood cancer treatment: impact on future health status--a review.

PURPOSE: To describe data on changes in body composition in childhood cancer survivors. Underlying mechanisms in development of obesity are addressed, in order to discuss intervention strategies. METHODS: A systematic literature search was undertaken with a number of search terms. RESULTS: Female survivors of ALL and brain tumours, especially if treated with cranial irradiation, showed a higher prevalence of obesity compared with the general population, while survivors of other malignancies had a higher prevalence of underweight. Influences of corticosteroid treatment and cytostatics on body composition are uncertain. Diminished physical activity, early adiposity rebound (<5 years of age) and/or hypothalamic involvement of tumour or treatment, and subsequent growth hormone deficiency, may play a role in the development of obesity in childhood cancer survivors. CONCLUSION: Longitudinal prospective studies in more extensive cohorts are necessary to estimate actual prevalence and facilitate the unravelling of the underlying mechanisms in change of body composition.

Late effects in adult survivors of childhood cancer: the need for life-long follow-up.

BACKGROUND: To assess health status and health-related quality of life (HRQoL) in childhood cancer survivors who were not involved in regular long-term follow-up. PATIENTS AND METHODS: One hundred and twenty-three long-term survivors, median age 33 (19-50) years, follow-up 27 (9-38) years, were recalled to the long-term follow-up clinic. Most of them were treated in the period 1970-1990. Late effects were graded using the Common Terminology Criteria for Adverse Events, version 3 (CTCAEv3). HRQoL was assessed by RAND-36. Socio-economic factors were compared with data from Statistics Netherlands (CBS). RESULTS: Grade 1-2 late effects were found in 54% of the survivors, grade 3-4 in 39%, two or more late effects in 70% and grade 2-4 previously unknown late effects in 33%. Survivors had significantly lower scores on RAND-36 compared with controls. CONCLUSIONS: As nearly 40% of these long-term childhood cancer survivors suffer from moderate to severe late effects and 33% had previously unknown late effects it is worthwhile recalling these patients to follow-up. Where and by whom this follow-up can best be done is still a question that needs to be answered.

Health-related quality of life and adverse late effects in adult (very) long-term childhood cancer survivors.

METHOD: The RAND-36 was used to assess HRQoL in all adult (> or =18 years) survivors who had attended the long-term follow-up clinic since 1995. The survivors were divided into two groups based on the length of follow-up: Group LF (long term follow-up, follow-up > or =20 years, n=129) and Group VLF (very long-term follow-up, follow-up >20 years, n=184). Data on diagnosis, treatment and complications were obtained from medical records. Late effects were graded using the CTCAEv3. RESULTS: The RAND-36 was completed by 313 (86.2%) out of 363 eligible patients. Except for higher scores on the subscale Bodily pain, LF patients did not differ significantly on the RAND-36 subscales from the population sample; VLF patients had significant lower scores on the subscales Physical functioning (P=0.003), Social functioning, Vitality and General health perception (P<0.001). Significantly more VLF patients (P<0.001) had severe (grade 3 and 4) late effects (47.8%) compared to LF patients (27.9%). Female gender and especially psycho-social late effects were inversely related to HRQoL. CONCLUSION: Childhood cancer survivors who were diagnosed more than 20 years ago have lower scores on the RAND 36, and have significantly more severe late effects than those diagnosed more recently. Patients with longer follow-up are more likely to become lost to follow-up. Time has come to establish new models of care for adult childhood cancer survivors, which are more flexible and appropriate to the needs of adult childhood cancer survivors.

Imminent ovarian failure in childhood cancer survivors.

The aim of this study was to investigate reproductive history and the prevalence of imminent ovarian failure (IOF) in female childhood cancer survivors. Reproductive history and ovarian function were evaluated by questionnaires (n=124) and by measurement of follicle stimulating hormone (FSH) and oestradiol (E2) levels (n=93). IOF was defined as FSH>10 IU/l or E2>0.28 nmol/l on day 3 of the menstrual cycle, or FSH>12.4 IU/l on day 7 of the pill-free interval. IOF was demonstrated in 22.6% of the participants and correlated with age at diagnosis (P<0.005) and age at study (P=0.036). IOF correlated inversely with methotrexate (P=0.046). The incidence of miscarriages (22.7%) and recurrent miscarriages (7.3%) was increased. The male/female (M/F) ratio of the offspring was decreased. In conclusion, female childhood cancer survivors are at risk for IOF. If pregnant, the risk of (recurrent) miscarriages is increased. The M/F ratio in the offspring is decreased.

Leukaemia incidence and survival in children and adolescents in Europe during 1978-1997. Report from the Automated Childhood Cancer Information System project.

Leukaemias constitute approximately one-third of cancers in children (age 0-14 years) and 10% in adolescents (age 15-19 years). Geographical patterns (1988-1997) and time trends (1978-1997) of incidence and survival from leukaemias in children (n=29,239) and adolescents (n=1929) were derived from the ACCIS database, including data from 62 cancer registries in 19 countries across Europe. The overall incidence rate of leukaemia in children was 44 per million person-years during 1988-1997. Lymphoid leukaemia (LL) accounted for 81%, acute non-lymphocytic leukaemia (ANLL) for 15%, chronic myeloid leukaemia (CML) for 1.5% and unspecified leukaemia for 1.3% of cases. Adjusted for sex and age, incidence of childhood LL was significantly lower in the East and higher in the North than in the British Isles. The overall incidence among adolescents was 22.6 per million person-years. The incidence of LL was rising in children (0.6% per year) and adolescents (1.9% per year). During 1988-1997 5-year survival of children with leukaemias was 73% (95% CI 72-74) and approximately 44% for infants and adolescents. Similar differences in survival between children and adolescents were observed for LL, much less so for ANLL. Survival differed between regions; prognosis was better in the North and West than the East. Remarkable improvements in survival occurred in most of the subgroups of patients defined by diagnostic subgroup, age, sex and geographic categories during the period 1978-1997. For children with ANLL most improvements in survival were observed in the 1990s.

Causes of death--other than progressive leukemia--in childhood acute lymphoblastic (ALL) and myeloid leukemia (AML): the Dutch Childhood Oncology Group experience.

We analyzed causes of death, other than resistant disease or relapse, in 875 children with acute lymphoblastic leukemia (ALL) and 229 with acute myeloid leukemia (AML), treated on three different Dutch Childhood Oncology Group (DCOG) ALL and three AML protocols. Overall, 23 (2.6%) ALL and 44 (19.2%) AML patients died. Early death (ED, before remission was reached) occurred in nine ALL (1%) and thirty AML (13.1%) patients, including three and ten deaths before treatment was initiated. Chemotherapy-related mortality in remission (CRM) occurred in nine ALL (1.1%) and eight AML (4.4%) patients. For ALL, both ED and CRM declined over time, although this was not statistically significant. For AML a decrease in ED was observed (from 26% to approximately 10%), but counter-balanced by an increase in CRM (from 3 to 8%), maybe related to the scheduling of intensification blocks in AML-92/94. Including transplant-related mortality, death in CR rates in AML increased from 3 to 15% in the last study. The main cause of ED was hemorrhage, often associated with hyperleucocytosis, and infection for CRM. We conclude that mortality dropped favorably in ALL, but not in AML. Especially for AML, effective but less toxic therapy and better supportive care guidelines need to be developed.

Inhibition of hemopoiesis in vitro by neuroblastoma-derived gangliosides.

Hemopoiesis is disturbed in bone marrow-involving cancers like leukemia and neuroblastoma. Shedding of gangliosides by tumor cells may contribute to this tumor-induced bone marrow suppression. We studied in vitro the inhibitory effects of murine neuroblastoma cells (Neuro-2a and C1300) and their gangliosides on hemopoiesis using normal murine hemopoietic progenitor colony-forming assays. Transwell cultured neuroblastoma cells showed a dose-dependent inhibition on hemopoiesis, indicating that a soluble factor was responsible for this effect. Furthermore, the supernatant of Neuro-2a cultured cells inhibited hemopoietic proliferation and differentiation. To determine whether the inhibitory effect was indeed due to shed gangliosides and not, for instance, caused by cytokines, the effect of DL-threo-1 -phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) on Neuro-2a cells was studied. DL-PDMP is a potent inhibitor of glucosylceramide synthase, resulting in inhibition of the synthesis and shedding of gangliosides. The initially observed inhibitory effect of supernatant of Neuro-2a cells was abrogated by culturing these cells for 3 days in the presence of 10 microM DL-PDMP. Moreover, gangliosides isolated from Neuro-2a cell membranes inhibited hemopoietic growth. To determine whether the described phenomena in vitro are a reflection of bone marrow suppression occurring in vivo, gangliosides isolated from plasma of neuroblastoma patients were tested for their effects on human hemopoietic progenitor colony-forming assays. These human neuroblastoma-derived gangliosides inhibited normal erythropoiesis (colony-forming unit-erythroid/burst-forming unit-erythroid) and myelopoiesis (colony-forming unit-granulocyte/macrophage) to a higher extent compared with gangliosides isolated from control plasma. Altogether these results suggest that gangliosides shed by neuroblastoma cells inhibit hemopoiesis and may contribute to the observed bone marrow depression in neuroblastoma patients.

Mobilized human CD34+ hematopoietic stem cells enhance tumor growth in a nonobese diabetic/severe combined immunodeficient mouse model of human non-Hodgkin's lymphoma.

Autologous peripheral blood stem cell mobilization is increasingly applied in the treatment of hematological malignancies. Despite the frequent clinical use in a setting of residual disease, it is not known whether mobilization of hematopoietic stem cells might facilitate tumor outgrowth in vivo. In the bone marrow, a bipotential precursor for hematopoietic and endothelial cells called hemangioblast exists. This hemangioblast, characterized by the expression of CD34 and vascular endothelial growth factor receptor (VEGFR)-2, is released from the bone marrow by mobilization and might be able to result in not only the generation of peripheral blood cells but vasculogenesis due to differentiation of the hemangioblast along the endothelial lineage [in addition to VEGFR-2 expression, angiopoietin-2 (ANG-2) expression can also be found in this stage]. New vessel formation in the tumor is critical for tumor growth. A xenotransplant model was established with 10 x 10(6) Daudi cells (non-Hodgkin's lymphoma) s.c. injected in the neck region of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, who were sublethally irradiated with 2 Gy. At day 10 after tumor inoculation, half of the mice were given 0.5 x 10(6) human CD34+ cells i.v., whereas the other half were given PBS i.v. The human CD34+ cells were obtained from leukapheresis samples of myeloma patients undergoing autologous peripheral blood stem cell mobilization. We compared tumor growth and human-specific VEGFR-2 and ANG-2 expression in the two groups. Tumor growth is enhanced 2-fold when mobilized hematopoietic human CD34+ cells are given compared with PBS controls (P = 0.004). In addition, the human-specific VEGFR-2 and ANG-2 reverse transcription-PCR was only positive in the tumors of mice i.v. injected with human CD34+ cells. This indicates that the injected human CD34+ cells home to the tumors and differentiate along the endothelial lineage. In the present study, we demonstrate that mobilized human CD34+ hematopoietic cells injected i.v. might facilitate the outgrowth of tumors in the setting of minimal residual disease. Malignant tumors are capable of incorporating human CD34+ hematopoietic cells. This study questions the safety of leukapheresis in patients with (residual) tumor and has important implications for further development of intensive chemotherapy protocols with autologous stem cell rescue.

A lack of neuroblastoma in Down syndrome: a study from 11 European countries.

An epidemiological investigation in 11 European countries comprising a total childhood population of 54.1 million children and using 8 separate data sources was conducted to evaluate the occurrence of neuroblastoma in Down syndrome (DS). No cases of DS were detected among 6724 infants and children with neuroblastoma, although more than five were expected. This highly significant result (P = 0.0045 according to the Poisson test) is consistent with data in the literature, which contains only two poorly detailed cases in epidemiological studies and one ganglioneuroma in a DS mosaic patient. Like other tumors, such as leukemias, testicular germ cell tumors and lymphomas are in excess in DS patients; the lack of neuroblastomas does not reflect a general decreased incidence of cancer but rather a specific underrepresentation of this precise tumor. S-100 b protein, the gene for which maps to the long arm of chromosome 21, (a) is overproduced in DS patients, (b) produces growth inhibition and differentiation of neural cells in vitro, (c) is abundant in good-prognosis neuroblastomas, and (d) has been shown to induce growth inhibition and differentiation and cell death in several human and murine neuroblastoma cell lines and could be responsible for this variation. Additional epidemiological and experimental studies are warranted to confirm our interpretation of these data.

The value of high-dose methotrexate-based neoadjuvant chemotherapy in malignant fibrous histiocytoma of bone.

PURPOSE: The value of high-dose methotrexate (HD-MTX)-based neoadjuvant chemotherapy was evaluated in patients with malignant fibrous histiocytoma (MFH) of bone. PATIENTS AND METHODS: Since 1977, MFH of bone was diagnosed in 17 patients (12 males and five females). Ten patients (59%), completed treatment with four courses of neoadjuvant chemotherapy as follows: HD-MTX, vincristine, doxorubicin, cyclophosphamide, bleomycin, and dactinomycin, or HD-MTX, 4(1)-epidoxorubicin, and carboplatin followed by local tumor resection (n = 3), curettage-cryosurgery (n = 2), amputation (n = 2), or tumor resection-endoprosthetic replacement or allograft (n = 3). After recovery from surgery, an additional six courses of polychemotherapy, including HD-MTX in nine patients, were administered. One patient changed to cisplatin- instead of HD-MTX-containing chemotherapy postoperatively. One additional patient received only adjuvant HD-MTX-containing polychemotherapy. Neoadjuvant MTX-containing chemotherapy was contraindicated in five patients (29%) due to age, cardiac insufficiency, or mental disorder. In one patient, neoadjuvant chemotherapy was cancelled after one course due to renal failure. Treatment consisted of amputation (n = 2), one course of chemotherapy and amputation (n = 1), hyperthermic isolated limb perfusion (HILP; n = 1), intraarterial chemotherapy, radiotherapy, and endoprosthetic replacement (n = 1), and a combination of chemotherapy and radiation treatment (n = 1). RESULTS: Five of six patients who received no HD-MTX-based neoadjuvant chemotherapy developed metastatic disease (83%); the median time to metastatic disease was 17 months (range, 3 to 44). In contrast, in 10 patients who completed treatment with HD-MTX-based neoadjuvant chemotherapy, with a mean follow-up time of 9.8 years (range, 2.3 to 15.7) and a median follow-up time of 10.8 years (range, 2.3 to 15.7) after diagnosis, no local recurrence or distant metastases were diagnosed (P < .005). CONCLUSION: Neoadjuvant HD-MTX-containing chemotherapy in addition to surgery has dramatically improved the prognosis of patients with MFH of bone.

High cure rate with a moderately intensive treatment regimen in non-high-risk childhood acute lymphoblastic leukemia. Results of protocol ALL VI from the Dutch Childhood Leukemia Study Group.

PURPOSE: Here we report the results of a nationwide cooperative study in the Netherlands on acute lymphoblastic leukemia (ALL) in children. The aim of the study was to improve the cure rate and to minimize side effects in a group of non-high-risk ALL patients, especially with regard to the CNS. A second aim was to study potential prognostic factors. METHODS: Children (age 0 to 15 years) with non-high-risk ALL (WBC count < 50 x 10(9)/L, no mediastinal mass, no B-cell phenotype, and no CNS involvement) were treated with a uniform protocol, ALL VI. The treatment protocol used 6-week induction regimen with three drugs (vincristine, dexamethasone, and asparaginase), three weekly doses of intravenous (IV) medium high-dose methotrexate (2 g/m2), and 2-year maintenance therapy that consisted of alternating 5-week periods of methotrexate and mercaptopurine and 2-week periods of vincristine and dexamethasone. In the first year of maintenance, triple intrathecal therapy was administered every 7 weeks. RESULTS: From December 1, 1984 until July 1, 1988, 291 children with ALL were diagnosed; 206 were categorized as non-high-risk (71%), and 190 were treated according to protocol ALL VI. At 8 years, the event-free survival (EFS) rate was 81% (SE = 3%) and survival rate 85% (SE = 2.9%); the median follow-up time was 7.3 years (range, 36 to 117 months). The CNS relapse rate was 1.1% (two of 184 patients who achieved a complete remission [CR]). The only factor found to be of negative prognostic importance in terms of EFS (P = .05) was a positive acid phosphatase reaction. CONCLUSION: For children with non-high-risk ALL, the combination of IV medium high-dose methotrexate (2 g/m2 times three), triple intrathecal therapy in the first year of maintenance treatment, and the use of dexamethasone for induction and pulses during maintenance treatment has proved to be highly effective, especially in the prevention of CNS relapse. A high cure rate was achieved without the use of anthracyclines, alkylating agents, and cranial irradiation.

Patient stratification based on prednisolone-vincristine-asparaginase resistance profiles in children with acute lymphoblastic leukemia.

PURPOSE: To confirm the prognostic value of a drug resistance profile combining prednisolone, vincristine, and l-asparaginase (PVA) cytotoxicity in an independent group of children with acute lymphoblastic leukemia (ALL) treated with a different protocol and analyzed at longer follow-up compared with our previous study of patients treated according to the Dutch Childhood Leukemia Study Group (DCLSG) ALL VII/VIII protocol. PATIENTS AND METHODS: Drug resistance profiles were determined in 202 children (aged 1 to 18 years) with newly diagnosed ALL who were treated according to the German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL)-92 protocol. RESULTS: At a median follow-up of 6.2 years (range, 4.1 to 9.3 years), the 5-year disease-free survival probability (pDFS) rate +/- SE was 69% +/- 7.0%, 83% +/- 4.4%, and 84% +/- 6.8% for patients with resistant (PVA score 7 to 9), intermediate-sensitive (PVA score 5 to 6), and sensitive (SPVA score 3 to 4) profiles, respectively (sensitive and intermediate-sensitive v resistant, P

High expression of Mcl-1 in ALK positive and negative anaplastic large cell lymphoma.

AIM: To gain more insight into the genes involved in the aetiology and pathogenesis of anaplastic large cell lymphoma (ALCL). METHODS: Serial analysis of gene expression (SAGE) was undertaken on the CD4+ALK+ (anaplastic lymphoma kinase positive) ALCL derived cell line Karpas299 and as comparison on CD4+ T cells. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed on five ALCL derived cell lines and 32 tissue samples to confirm the SAGE data. RESULTS: High expression of Mcl-1 was seen in the Karpas299 cell line, whereas the two other antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-X(L), were not detected in the SAGE library. Quantitative RT-PCR confirmed the high expression of Mcl-1 mRNA and low expression of Bcl-2 and Bcl-X(L) in Karpas299 and in four other ALCL cell lines. To expand on these initial observations, primary tissue samples were analysed for Mcl-1, Bcl-X(L), and Bcl-2 by immunohistochemistry. All 23 ALK+ and nine ALK- ALCL cases were positive for Mcl-1. Bcl-2 and Bcl-X(L) were expressed infrequently in ALK+ ALCL cases, but were present in a higher proportion of ALK- ALCL cases. CONCLUSION: The consistent high expression of Mcl-1 in ALK+ and ALK- ALCL suggests that Mcl-1 is the main antiapoptotic protein in this disease. The high frequency of Mcl-1, Bcl-2, and Bcl-X(L) positive ALCL cases in the ALK- group compared with the ALK+ group indicates that ALK induced STAT3 activation is not the main regulatory pathway in ALCL.

Long-term follow-up of Dutch Childhood Leukemia Study Group (DCLSG) protocols for children with acute lymphoblastic leukemia, 1984-1991.

Here we report the long-term results of the DCLSG protocols ALL-6 and -7 with special emphasis on the incidence of CNS relapse after treatment without cranial irradiation. In DCLSG protocol ALL-6 (1984-1988), designed for patients with ALL non-high risk (ALL-NHR) (WBC <50 x 10(9)/l, no mediastinal mass, no B cell phenotype and no CNS involvement at diagnosis, comprising 71% of all ALL patients), CNS prophylaxis consisted of a combination of three methods of chemotherapeutic CNS prophylaxis (the use of dexamethasone during induction and maintenance therapy, i.v. medium dose methotrexate and prolonged administration of intrathecal triple therapy). Total duration of treatment: 116 weeks. 190 patients were enrolled in the study. At 10 years, the EFS rate for all patients is 81.5 +/- 2.8%, the survival rate 84.8 +/- 2.7%, and the cumulative incidence of isolated CNS relapse 1.1 +/- 0.8%. The 10-year survival rate for the 139/190 (73.1%) patients with standard risk non-T lineage ALL according to the NCI risk criteria is 80.5 +/- 3.4%. DCLSG protocol-7 was identical to the intensive ALL-BFM-86 protocol, but cranial irradiation was restricted to patients with initial CNS involvement. Patients were stratified into three risk groups (SRG, RG and EG). Treatment duration was 18 months. 218 patients were enrolled in the study. At 10 years, the EFS rate for all patients is 63.4 +/- 3.3%, the survival rate 76.4 +/- 3.0%, the 5-year cumulative incidence of isolated CNS relapse 5.7 +/- 1.8%. The EFS rate at 10 years of the 127/218 (58.3%) patients with standard risk non-T-lineage ALL according to the NCI risk criteria was 67.9 +/- 4.3%, which is not significantly different from the results achieved in this category of patients with the moderately intensive treatment according to protocol ALL-6 (logrank P = 0.17). These DCLSG studies indicate that omission of cranial irradiation does not jeopardize the overall good results.

Precursor-B-ALL with D(H)-J(H) gene rearrangements have an immature immunogenotype with a high frequency of oligoclonality and hyperdiploidy of chromosome 14.

The IGH gene configuration was investigated in 97 childhood precursor-B-ALL patients at initial diagnosis. Rearrangements were found by Southern blotting in all but three patients (97%) and in 30 cases (31%) we observed oligoclonal IGH gene rearrangements. Heteroduplex PCR analysis revealed at least one clonal PCR product in all Southern blot-positive cases. In 89 patients (92%) complete V(D)J rearrangements were found, while incomplete D(H)-J(H) rearrangements occurred in only 21 patients (22%). In 5% of cases the D(H)-J(H) rearrangements were the sole IGH gene rearrangements. Sequence analysis of the 31 identified incomplete rearrangements revealed preferential usage of segments from the D(H)2, D(H)3 and D(H)7 families (78%). While D(H)2 and D(H)3 gene rearrangements occur frequently in normal B cells and B cell precursors, the relatively frequent usage of D(H)7-27 (19%) in precursor-B-ALL patients is suggestive of leukemic transformation during prenatal lymphopoiesis. Among J(H) gene segments in the incomplete D(H)-J(H) rearrangements, the J(H)6 segment was significantly overrepresented (61%). This observation together with the predominant usage of the most upstream D(H) genes indicates that many of the identified clonal D(H)-J(H) gene rearrangements in precursor-B-ALL probably represent secondary recombinations, having deleted pre-existing D(H)-J(H) joinings. The patients with incomplete D(H)-J(H) gene rearrangements were frequently characterized by hyperdiploid karyotype with additional copies of chromosome 14 and/or by IGH oligoclonality. The presence of incomplete D(H)-J(H) joinings was also significantly associated with a less mature immunogenotype: overrepresentation of V(H)6-1 gene segment usage, absence of biallelic TCRD deletions, and low frequency of TCRG gene rearrangements. This immature immunogenotype of precursor-B-ALL with incomplete IGH gene rearrangements was not associated with more aggressive disease.