Programmed death (PD)-1/PD-ligand 1 blockade mediates antiangiogenic effects by tumor-derived CXCL10/11 as a potential predictive biomarker.

Immune checkpoint inhibitor (ICI) programmed death (PD)-1/PD-ligand 1 (PD-L1) blockade has been approved for various cancers. However, the underlying antitumor mechanisms mediated by ICIs and the predictive biomarkers remain unclear. We report the effects of anti-PD-L1/PD-1 Ab in tumor angiogenesis. In syngeneic mouse models, anti-PD-L1 Ab inhibited tumor angiogenesis and induces net-like hypoxia only in ICI-sensitive cell lines. In tumor tissue and serum of ICI-sensitive cell line-bearing mice, interferon-γ (IFN-γ) inducible angiostatic chemokines CXCL10/11 were upregulated by PD-L1 blockade. In vitro, CXCL10/11 gene upregulation by IFN-γ stimulation in tumor cell lines correlated with the sensitivity of PD-L1 blockade. The CXCL10/11 receptor CXCR3-neutralizing Ab or CXCL11 silencing in tumor cells inhibited the antiangiogenic effect of PD-L1 blockade in vivo. In pretreatment serum of lung carcinoma patients receiving anti-PD-1 Ab, the concentration of CXCL10/11 significantly correlated with the clinical outcome. Our results indicate the antiangiogenic function of PD-1/PD-L1 blockade and identify tumor-derived CXCL10/11 as a potential circulating biomarker of therapeutic sensitivity.

Hypocellular acute myeloid leukemia treated with bone marrow transplantation.

Hypocellular acute myeloid leukemia (AML) mainly occurs in elderly patients, and is extremely rare in childhood. There is still no established treatment for hypocellular AML. We report the case of an 11-year-old boy with hypocellular AML who was treated successfully with allogenic bone marrow transplantation (allo-BMT). He presented with fever, pallor and pancytopenia. Bone marrow aspiration and biopsy confirmed a diagnosis of hypocellular AML. Although low-dose cytarabine induced reduction of blasts, it did not lead to complete remission. He subsequently received myeloablative conditioning and allo-BMT. Graft-versus-host disease (GVHD) prophylaxis included short-course methotrexate and cyclosporine. Neutrophil engraftment (>5 × 108 /L) and platelet recovery (>10 × 1010 /L) were achieved on days 13 and 27, respectively. He developed acute GVHD of the skin (grade 2), which responded well to treatment with prednisolone. He has remained in complete remission for 5 years since allo-BMT. We consider allo-BMT to be feasible for children with hypocellular AML.

Susceptibility to 6-MP toxicity conferred by a NUDT15 variant in Japanese children with acute lymphoblastic leukaemia.

Genotyping of TPMT prior to 6-mercaptopurine (6-MP) administration in acute lymphoblastic leukaemia (ALL) patients has been integrated into clinical practice in some populations of European ancestry. However, the comparable rates of 6-MP myelotoxicity, but rarity of TPMT variants, in Asians suggest that major determinants have yet to be discovered in this population. We genotyped 92 Japanese paediatric ALL patients for NUDT15 rs116855232, a 6-MP toxicity-related locus discovered in Asians. Logistic regression and survival analysis were used to evaluate its association with leucopenia, hepatotoxicity, 6-MP dose reduction, therapy interruption and event-free survival. The allele frequency of rs116855232 was 0·16, and leucopenia was more common in carriers of the T allele (odds ratio, 7·20; 95% confidence interval, 2·49-20·80; P = 2·7 × 10(-4) ). As leucopenia results in 6-MP dose reduction, we observed average doses during maintenance therapy of 40·7, 29·3 and 8·8 mg/m(2) for patients with CC, CT and TT genotypes, respectively (P < 0·001). Hepatotoxicity was observed only in CC genotype patients. Event-free survival did not significantly differ by NUDT15 genotype. rs116855232 is an important determinant of 6-MP myelotoxicity in Japanese children with ALL and may represent the most robust toxicity-related locus in Asians to date. Considerations for clinical application may be warranted.

High-dose chemotherapy followed by autologous peripheral blood stem cell transplantation for recurrent primary mediastinal malignant germ cell tumor: a case report.

A 15-yr-old boy presented with an anterior mediastinal mass, multiple lung metastases and obstruction of the left brachiocephalic vein, the superior vena cava and the subclavian vein. Tumor biopsy by CT guidance confirmed a diagnosis of GCT. Five courses of BEP therapy were performed, and CT of the chest revealed reduction in the anterior mediastinal mass and disappearance of the multiple lung metastases. We performed the anterior mediastinal mass extraction followed by adjuvant chemotherapy consisting of ICE and TIP. However, the AFP levels became elevated soon after. Abnormal accumulation was observed in the right upper lung by DW-MRI. After the operation, two courses of TI chemotherapy and two courses of HDCT followed by auto-PBSCT were performed. He was complicated with auditory disorder and renal dysfunction. Although HDCT followed by auto-PBSCT was effective for the relapsed primary mediastinal GCT, a treatment strategy avoiding late complications is warranted.

[Efficacy of chemotherapy combined with bortezomib for two cases of relapsed/refractory acute lymphoblastic leukemia].

Bortezomib (BZM), a proteasome inhibitor, was recently reported to be effective against acute lymphoblastic leukemia (ALL). We report two cases of relapsed/refractory ALL, who were treated with BZM (1.3 mg/m2/dose, 2 doses/week for 2 weeks) in combination with vincristine, doxorubicin, dexamethasone, and L-asparaginase (L-ASP). The first patient was a 16-year-old girl who developed a bone marrow relapse 8 months after the initial diagnosis during consolidation chemotherapy. She received BZM-combined chemotherapy without L-ASP considering her previous history of an allergic reaction to L-ASP. The BZM-combined regimen was discontinued due to interstitial pneumonia development on day 13, and the interstitial pneumonia was successfully treated with steroid pulse therapy. Although her elevated serum LDH transiently normalized on day 16, blasts in peripheral blood did not disappear, and she died of leukemia without achieving remission. The second patient was a 17-year-old girl who developed a third bone marrow relapse after cord blood transplantation. She was given the same BZM combined regimen. Although the BZM-combined regimen was discontinued due to acute pancreatitis development on day 12, complete remission without platelet recovery was confirmed on day 62. Our experience suggests not only the effectiveness of BZM-combined chemotherapy but also the importance of controlling its toxicities when administered as a salvage therapy for advanced ALL patients.

T-cell large granular lymphocyte leukemia in a child with anemia and Crohn's disease.

T-LGL leukemia has been rarely reported in children. We report a child with T-LGL leukemia who presented with anemia and went on to develop Crohn's disease. Although prednisolone treatment proved effective in the treatment of anemia, large granular lymphocyte counts increased as the doses were tapered. T-cell rearrangement studies revealed a clonal rearrangement of the TCR Vß/jß2 gene. Concurrently, the patient developed severe diarrhea. Inflammatory changes across the upper and lower intestines led to the diagnosis of Crohn's disease. This case highlights that T-LGL leukemia could be occurred in children. Flow cytometry and/or T-cell gene rearrangement studies are recommend in patients of anemia and various kind of autoimmune diseases including Crohn's disease, even in children.

Altered cellular immunity in transgenic mice with T cell-specific expression of human D4-guanine diphosphate-dissociation inhibitor (D4-GDI).

D4-GDI, a Rho guanosine diphosphate (GDP) dissociation inhibitor, is preferentially expressed in hematopoietic tissues and binds to a small GTP-binding protein, Rho, and inhibits GDP dissociation from Rho. We identified point mutations in the D4-GDI gene in human leukemic cells. We therefore investigated the functions of D4-GDI and mutated D4-GDI in T cells. Transgenic mice (Tg) harboring human wild-type and mutant D4-GDI transgenes driven by the lck promoter were generated. Cellular immunity responses against cytozoic pathogens were examined. The cytoskeletal organization in the CD3+T cells and the proliferation of splenocytes by Con A were investigated in both Tg and littermates (LMs). Granuloma formation by bacille Calmette-Guerin was impaired in the wild-type D4-GDI Tg. On the other hand, the number of granulomas of the mutated D4-Tg was significantly higher. Infection with Listeria was more rapidly fatal to wild-type D4-GDI Tg than to LMs, while the survival of mutated D4-GDI Tg was prolonged. The CD3+T cells in wild-type D4-GDI Tg showed an impairment in the formation of stress fibers on anti-CD3 antibody-coated plates, whereas the cytoskeletal organization in CD3+T cells of the mutated D4-GDI Tg was augmented. The proliferation of splenocytes after Con A stimulation was higher in the mutated D4-GDI Tg than in the LMs. D4-GDI may have important functions, such as induction of T cell migration, adhesion and/or proliferation in inflammatory foci, in cellular immunity responses to cytozoic pathogens.

Mutated D4-guanine diphosphate-dissociation inhibitor is found in human leukemic cells and promotes leukemic cell invasion.

OBJECTIVE: Rho GTPase may be involved in human cancer invasion via the augmentation of cell motility and adhesion. We report on two point mutations of the D4-guanine diphosphate (GDP)-dissociation inhibitor (GDI) gene, one of the Rho-GDIs, which were found in a human leukemic cell line, Reh, and the mutated D4-GDI functions as an accelerator of leukemic cell invasion. MATERIAL AND METHODS: We investigated the altered activity of GDP dissociation by mutated (mt) D4-GDI and the functions of this mt and wild-type (wt) D4-GDI in invasion. The mice inoculated with wt or mt D4-GDI vector-transfected Raji cells were observed and examined pathologically. Adhesiveness and cell motility of wt or mt D4-GDI vector-transfected Raji cells were examined. Finally, it was examined whether Rho activation was changed by mutation of D4-GDI under the condition of Rho-GDI knockdown. RESULTS: Two point mutations of the D4-GDI gene were found in Reh cells. The region of mutations is conserved among members of the Rho-GDI family at the amino acid level. D4-GDI with two mutations (V68L and V69A) functioned in a dominant negative manner in the inhibition of GDP dissociation from Rho. Severe combined immune-deficient mice inoculated with Raji cells developed hemiparalysis. The Raji cells were present in bone marrow and peripheral blood, and hepatic invasion was observed in 20% of the mice. Mice inoculated with wt D4-GDI vector-transfected Raji cells (wt D4) showed later paralysis and none developed hepatic invasion. Mice inoculated with mt D4-GDI-transfected Raji cells (mt D4) showed a 5-day reduction in the time to paraplegia and death. In addition, hepatic invasion was evident in 80% of mice transplanted with mt D4 cells. There were no differences in growth rates and amounts of guanine triphosphate (GTP)-bound Rho, cdc42, or Rac among all clones, however, GTP-bound Rho in mt D4 clone with short hairpin RNA (shRNA) vector for Rho-GDI knockdown was increased compared with wt D4 clone with shRNA vector for Rho-GDI knockdown. The mt D4 cells showed an augmentation of adhesiveness and cell motility. On the other hand, wt D4 cells showed a decreased ability of cell motility. CONCLUSION: These results suggest the mutated D4-GDI functions as a dominant negative molecule against the wt D4-GDI and accelerates invasion via regulation of cytoskeletal machinery.

Effects of sodium in hydration solution on plasma methotrexate concentrations following high-dose methotrexate in children with acute lymphoblastic leukemia.

PURPOSE: To test whether a higher sodium dose in the hydration solution may facilitate faster methotrexate (MTX) elimination as compared with a lower sodium dose following high-dose MTX (HDMTX) treatment. METHODS: Intravenous solutions with alternate doses of sodium (regimen A 70 mEq/l, regimen B 100 mEq/l) were given to 30 children with acute lymphoblastic leukemia in two courses of HDMTX in a randomized crossover fashion. The plasma MTX concentrations every 24 h from the beginning of MTX administration and the adverse events associated with HDMTX were compared between the two hydration regimens. RESULTS: The plasma MTX concentrations were similar in the two hydration regimens at 24 h (A 50.9+/-7.4 vs B 40.9+/-5.4 microM, means+/- SE, P=0.17), but was significantly lower in regimen B at 48 and 72 h (A 0.65+/-0.17 vs B 0.27+/-0.03 microM, P=0.04; and A 0.14+/-0.03 vs B 0.05+/-0.01 microM, P=0.003). The time during which MTX plasma concentrations exceeded 0.1 microM was significantly longer in regimen A than in regimen B (A 3.83+/-0.18 vs B 3.13+/-0.06 days, P=0.001). The incidences of adverse events were similar between the two regimens ( P=0.78), and severe adverse events were not seen in either regimen. CONCLUSIONS: Hydration with a higher sodium dose facilitated faster MTX elimination following HDMTX. Sodium may have a beneficial effect on MTX-induced nephrotoxicity.

A pediatric case of secondary leukemia associated with t(16;21)(q24;q22) exhibiting the chimeric AML1-MTG16 gene.

A chimeric gene, AML1-MTG16, showing high homology to AML1-MTG8, was recently identified in adult leukemic patients with the abnormal karyotype t(16;21)(q24;q22). We recently saw a child patient of 11 years of age who developed acute myelogenous leukemia with the karyotype t(16;21)(q24;q22), 11 months after autologous peripheral blood stem-cell transplantation (PBSCT) for acute promyelocytic leukemia with karyotype t(15;17)(q22;q11). The reciprocal translocation was localized by fluorescence in situ hybridization (FISH) analysis, reverse transcription polymerase chain reaction (RT-PCR), and Southern blot analysis of bone marrow blood cells and peripheral blood cells. FISH analysis identified a reciprocal translocation between chromosomes 16 and 21. RT-PCR analysis identified expression of the chimeric gene AML1-MTG16. Southern blot analysis revealed a breakpoint occurring at a 1.4 kb Eco RI fragment between exons 3 and 4 of MTG16. The breakpoint is within the same region as that of secondary leukemias, which has been reported previously. This case suggests the possibility that the region of the breakpoint of MTG16 is a characteristic of secondary leukemia.

Various applications of direct PCR using blood samples.

Samples of blood or other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), meaning that isolation of DNA, involving multiple labor-intensive steps, is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances. Using this reagent cocktail, DNA from various targets can be efficiently amplified directly from various forms of blood samples without DNA isolation. 1. DNA sequences within the beta-globin gene could be amplified directly from human blood samples treated with various anticoagulants. Either fresh blood or blood samples stored frozen for up to 4 years could be used for PCR. 2. DNA sequences of up to 2056 bp within the beta-globin gene could be amplified directly from human blood samples. 3. Human chromosomal and mitochondrial DNA from different individuals could be amplified directly from blood samples. 4. Low titers of hepatitis B virus could be amplified directly from human blood samples. 5. DNA could be amplified directly from various target sequences using dried blood in a PCR tube or on a filter paper. 6. Transgenes could be detected directly in blood samples from transgenic mice.

Methylenetetrahydrofolate reductase gene haplotypes affect toxicity during maintenance therapy for childhood acute lymphoblastic leukemia in Japanese patients.

Abstract The aim of this study was to investigate the influence of daily 6-mercaptopurine (6-MP) and low-dose weekly methotrexate (MTX) combination treatment and methylenetetrahydrofolate reductase (MTHFR) haplotypes on toxicity during maintenance therapy in Japanese childhood acute lymphoblastic leukemia (ALL). We retrospectively analyzed the MTHFR C677T and A1298C polymorphisms and influence of haplotypes on toxicity in 73 patients. Patients with the MTHFR 677TT and 677CT + 1298AC were associated with severe liver toxicity (p = 0.014, odds ratio [OR] = 3.82, 95% confidence interval [CI] = 1.27-11.46) and more rapid onset of liver toxicity (p = 0.010). Patients with MTHFR 677TT and 677CT + 1298AC were associated with lower frequency of 6-MP and MTX dose reduction due to leukopenia (p < 0.05). No difference was observed in average drug doses in the MTHFR genotypes. In conclusion, the MTHFR C677T and A1298C haplotypes might be useful for monitoring adverse effects in childhood ALL maintenance therapy in Japanese patients.

The activity of the inosine triphosphate pyrophosphatase affects toxicity of 6-mercaptopurine during maintenance therapy for acute lymphoblastic leukemia in Japanese children.

The association between inosine triphosphate pyrophosphatase (ITPA) activity and toxicity of 6-mercaptopurine (6-MP) was retrospectively evaluated in 65 Japanese children with acute lymphoblastic leukemia (ALL). Patients with an ITPA activity of less than 126 µmol/h/gHb presented with hepatotoxicity more frequently than those with higher ITPA activity (p<0.01). The average 6-MP dose during maintenance therapy administered to two patients with the ITPA deficiency was lower than that given to the other patients. Measuring ITPA activity is important for ensuring the safety of maintenance therapy for Asians with ALL because thiopurine S-methyl transferase mutations are rare in the Asian population.

Establishment of a novel childhood acute myeloid leukaemia cell line, KOPM-88, containing partial tandem duplication of the MLL gene and an in vivo model for childhood acute myeloid leukaemia using NOD/SCID mice.

MLL gene rearrangement is common in both adult and childhood acute myeloid leukaemia (AML), and its role in oncogenesis has been investigated. While over 50 translocated-partner genes have been identified so far, few studies have detailed the molecular mechanism of partial tandem duplication (PTD) of the MLL gene. The prognostic impact and contribution to leukaemogenesis of MLL-PTD, especially in childhood cases, remain unknown. We have established a novel cell line containing MLL-PTD derived from an 11-year-old patient with AML and designated as KOPM-88. KOPM-88 cells exhibited certain characteristics associated with the myeloid lineage including abundant primary granules in the cytoplasm and the expression of myeloperoxidase. The cell growth of KOPM-88 was cytokine independent but was accelerated by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. MLL-PTD of exon 2 to exon 6 and exon 2 to exon 8 was revealed using Southern blotting, fluorescence in situ hybridisation, and reverse transcription polymerase chain reaction/DNA sequencing. Furthermore, non-obese diabetic/severe combined immunodeficient mice inoculated with KOPM-88 cells exhibited leukaemic infiltrations in the bone marrow and hemiparalysis because of compression myelopathy. This is the first report of an in vivo animal model exhibiting the systemic involvement of childhood AML containing MLL-PTD. KOPM-88 cells and our murine model may be useful for investigating the pathogenesis of childhood AML associated with MLL gene rearrangement.

Exogenous expression of heat shock protein 90kDa retards the cell cycle and impairs the heat shock response.

The 90-kDa heat shock protein, HSP90, is an abundant molecular chaperone which functions in cellular homeostasis in prokaryotes and eukaryotes. It is well known that HSP90 plays a critical and indispensable role in regulating cell growth through modulations of various signal transduction pathways, but its roles in cell cycle control are not so well known. We transferred human HSP90 (wild-type or mutated types) expression vectors into NIH-3T3 cells in order to study certain functions of HSP90 in the cell cycle and cell growth under physiological conditions. We found that the exogenous expression of HSP90 (wild-type) induced a decrease in cell growth via retardation of the G1/S transition. The inhibition of cell growth was caused by reduced expressions of cyclin D3 and cyclin A mRNA and protein. On the other hand, no stable transfectants with the three types of mutated HSP90 were obtained. Unexpectedly, exogenous HSP90 expression impaired the heat shock response by inhibiting both heat shock transcription factor 1(HSF1) activation and transportation of HSF1 into the nucleus. The HSF1 function was disrupted by the direct association between HSF1 and exogenous HSP90, which was present as a monomer. These results reveal important roles of HSP90 in cell cycle control and in the stress response of nontransformed cells.

Tumor angiogenesis in the bone marrow of multiple myeloma patients and its alteration by thalidomide treatment.

Angiogenesis in solid tumors is important to tumor growth, invasion and metastasis. Recently, it has been suggested that angiogenesis plays a certain role in the development of hematopoietic malignancies, including leukemia and multiple myeloma. We evaluated tumor angiogenesis in the bone marrow (BM) of multiple myeloma (MM) patients by calculating microvessel density (MVD) in needle-biopsy specimens obtained from 51 cases of untreated MM or monoclonal gammopathy of undetermined significance (MGUS). The MVD in the BM of donors for transplantation and patients with non-hematological diseases was calculated as a control. There was an obvious increase in MVD in the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.