bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Chemo Sensitivity Index for Melphalan Based on Gene Expression Profiling (GEP).
Authors: Dahlgaard Jesper O. , Dybkaer Karen , Johnsen Hans Erik , Knudsen Steen
Published: October 2019

Multiple Myeloma (MM) is a lethal hematological malignancy with an incidence of 40–60 per 1.000.000 per year. Over the last decade, the most significant therapeutic improvement has been the introduction of high-dose Melphalan therapy in combination with autologous stem cell support. This strategy has led to an increase in survival from a median of approximately 3 years till now >6 years also in Denmark (unpublished data from Nordic Myeloma Study Group, NMSG #7/98). Despite this important increase in survival, substantial variability still persists among patients in response to high-dose Melphalan. Clinical and genetic heterogeneity is a characteristic feature of MM, and new methodological approaches, such as global gene expression profiling (GEP), are valuable tools in order to link clinical endpoints, such as response to treatment and survival, with the molecular and genetic heterogeneity of the disease (Shaughnessy, J. et al., Blood98, 217–223, 2001). Here we present a bioinformatic analysis of the clinical impact of a novel chemo sensitivity index based on GEP of 1) a panel of sixty cell lines (NCI60) that subsequently have been exposed to a range of anti-cancer agents, including Melphalan, and, 2) a retrospective validation of our method from lung, brain and lymphoma patients. Figure 1, presents a retrospective leave-one-out cross-validation of the prediction whether a patient will respond to the treatment given. Lung- and brain cancer patients were treated with Cisplatin, and the predicted sensitivity or resistance to this drug was used to group patients (hazard ratio 2.9, 95% CI 0.7–11). Lymphoma patients were treated with Vincristine and Adriamycine, and the patients were grouped according to their predicted sensitivity or resistance to these drugs. Patient and raw DNA chip data were obtained from: Beer et al, Nature Medicine 8(8):816–824; Pomeroy et al, Nature 415(6870):436–442; Shipp et al, Nature Medicine 8(1):68–74. For each patient, the method may also allow for a quantitative prediction of drug sensitivity towards a range of drugs including Melphalan. Figure 2 shows the drug sensitivity profile for two selected Multiple Myeloma patients (patient #2 and #9 from Agnelli L et al. Haematologica. 2007 Jan; 92(1):56–65). We are currently collecting chip data from a cohort of 70 newly diagnosed biobanked bone marrow samples from MM patients treated with high-dose Melphalan in NMSG trial #9/99 in order to further confirm these results in MM. These data are subsequently being validated using an independent population of bone marrow samples from MM patients also treated with high-dose Melphalan. We demonstrate that GEP can provide new information concerning resistance towards Melphalan and a range of anti-cancer drugs, separately or in combination (multi drug regimen), which may be clinically useful, and serve as a guide for the future development of individualized treatment strategies.

Figure Figure
The Study of Bortezomib Alone or in Combination with Harringtonine or Arsenic Trioxide on Proliferation and Apoptosis of Multidrug Resistant Leukemic Cells.
Authors: Meng Fan Yi , Cai Yan Xia , Sun Qi Xin , Fu Yun Bi , Li Li
Published: October 2019

To investigate the effect of bortezomib alone or combined with harringtonine (HT) or arsenic trioxide (As2O3) on the proliferation activity and apoptosis of HL60/ADM cell line and refractory/relapse acute leukemia primary cell. HL60/ADM cells or refractory/relapse acute leukemia primary cells were incubated with bortezomib at different doses alone and in combination with HT or As2O3. The proliferation activity was abserved by MTT assay, cell apoptosis was studied by fluorescence microscopy and flow cytometry. Intracellular concentration of daunorubicin (DNR) was determined by flow cytometry. In bortezomib-treated tumor cells, inhibition rate enhanced and apoptotic cells increased in mumber time- and dose-dependently. 40nM Bortezomib can best inhibit the proliferation activity of HL60/ADM cells when incubated for 48 hours. 15uM As2O3 or 752nM HT combined with different doses of bortezomib can inhibit the proliferation and induce the apoptosis of HL60/ADM cells, The dual inhibition of As2O3 plus bortezomib or HT plus bortezomib may present a superior anticancer efficancy to either inhibition of the drugs above alone (P<0.05;<0.01). Bortezomib (10nM) can markedly enhance the intraceflular accumulation of DNR in HL60/ADM cells(P=0.000). Bortezomib can inhibits proliferation and induces apoptosis of HL60/ADM cells and refactory/ relapse acute leukemic primary cells. When combined with HT or As2O3 appear to have synergistic effects, and the mechanisms might be associated with the ability of reversing multidrug resisitant of Bortezomib.
RARα-Specific Retinoid Am-80 Prevents SDF-1-Mediated Chemotaxis in CXCR4 Expressing Tumor Cells.
Authors: Matsumoto Taichi , Jimi Shiro , Suzumiya Junji , Hara Shuji , Tamura Kazuo
Published: October 2019

Chemokine (C-X-C motif) receptor 4 (CXCR4) plays an important role on hematogenous metastasis of tumor cells to the organs comprising constitutively stromal cell-derived facter-1 (SDF-1). Retinoids have been utilized as a drug for treatment of acute promyelocytic leukemia (APL), in which tumor cells give rise to differentiation and cell death by activation of retinoic acid receptors (RARs). However, the effects of retinoid on chemokine-mediated cell motility are totally unknown. In this study, we examined the effects of retinoids, including all-trans retinoic acid (ATRA) and RARα-specific retinoid Am-80, on CXCR4-mediated chemotactic activity using five myeloid and lymphoid leukemia cells (HL-60, K562, MOLT-3, Jurkat, and U266B1). When cells were incubated with ATRA or Am-80 at doses up to 10 mM for 3 days, comparable effects were found; cytotoxic effect occurred only in HL-60, and growth inhibition was albeit detected in all of other cells. The extents of cell surface CXCR4 in all of the cells detected by flow cytometric analysis tended to be correlated with SDF-1-induced migration activity. Cell surface CXCR4 was slightly decreased by ATRA treatment, while it was significantly decreased by Am-80 treatment, and the alterations were in time- and dose-dependent fashions. In terms of CXCR4 expression, Jurkat was the most sensitive cells. Cell migration in response to SDF-1, was monitored by a modified Boyden chamber method. SDF-1 induced significant chemotaxis in HL-60, MOLT-3 and Jurkat cells, but did not in K562 and U266B1 cells. After treatment with the retinoids, HL-60 was only a cell type increasing random cell motility, however in retinoids-pretreated MOLT-3 and Jurkat cells, no such effect was found, and therefore suggesting that up-regulation of the motility in HL-60 is maybe due to a response by dyeing cells. However, in a case of pretreatment with not ATRA but Am-80, SDF-1-induced chemotaxis of MOLT-3 and Jurkat cells was dose-dependently repressed in accordance with decrease in cell surface CXCR4 expression. This was also confirmed by a time-laps horizontal migration system, indicated that pseudopodia formation accompanied with F-actin rearrangement by SDF-1 was strongly inhibited by Am-80-pretreatment. These results indicate that RARα-specific retinoid Am-80 may induce not only growth inhibition, but also prevention of SDF-1/CXCR4-mediated hematogenous metastasis in CXCR4 expressing tumor cells.
Pharmacogenetic Study of Methotrexate and 6-Mercaptopurine Related Genes in Korean Pediatric Patients with Acute Lymphoblastic Leukemia.
Authors: Kang Hyoung Jin , Seo Young-Jin , Lee Chang Jin , Kim Hyery , Jang Mi Kyung , Koh Sang Hyeok , Oh Yongtaek , Han Byoung-Don , Kim Chul Woo , Kim Eun Kyung , Shin Hee Young , Ahn Hyo Seop
Published: October 2019

The cure rate of pediatric acute lymphoblastic leukemia (ALL) has dramatically improved after the introduction of maintenance therapy. But many Korean patients could not tolerate the planned dose of methotrexate (MTX) and 6-mercaptopurine (MP) from Western protocols. Recently, tailored therapy based on individual pharmacogenetic background is introduced to improve the survival with the reduction of complication. To establish the basis for the tailored therapy, genes associated with MTX and MP metabolism were analyzed in Korean children with ALL. The dose percent to the planned dose of MTX (20 mg/m2) and MP (50 mg/m2) at final maintenance cycle were analyzed with the 7 polymorphism site (MTHFR 677 C>T, MTHFR 1298 A>C, RFC 80 G>A, TYMS enhancer repeat, TPMT 238 G>C, TPMT 460 G>A, TPMT 719 A>G) in 96 ALL patient using TotalPlex PCR method (except TYMS). The median dose percent of MTX and MP of all patients were 57.5% and 49%, respectively. Only 31/96 and 22/96 patients tolerated more than 75% of planned MTX and MP doses, respectively. Variant TPMT was found in 6 patients (one *1/*3B and five *1/*3C). The mean dose percent of MP in patients with variant TPMT (33.2%) was lower than that of others (53.5%), significantly (P=0.04). MTHFR 677, MTHFR 1298, RFC 80, and TYMS enhancer repeat were not associated with the dose percent of MTX. But the dose percent of MTX was correlated with the dose percent of MP significantly (R2=0.64, P=0.00). The 5 year event free survival of all patients was 93.7%. Although 16/96 patients received less than 25% of full MP dose, there was no event in these patients. Dose reduction of MP is recommended for the patients with 3 major variant types of TPMT but many others with wild type could not tolerate the planned dose of MP. Study of novel polymorphism is necessary for the basis of tailored therapy in Korean pediatric ALL patients.
Carbonyl-Substituted Titanocenes as New Cytostatic Agents Against Lymphoma and Leukemia in Childhood.
Authors: Selig Anja , Wagner Laura , Winkler Iris , Lauterbach Thorsten , Gansaeuer Andreas , Prokop Aram
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most common malignant disease in childhood. Despite a relatively good prognosis approximately one fourth of the patients suffers from relapse and, consequently, a worse prognosis. Only 40% of these children with relapsed ALL will survive. Patients who fail to achieve a complete remission in chemotherapy do not survive. This explains the need for new compounds that may overcome resistance against clinically employed cytostatic drugs. In the present study we examined novel titan-containing agents concerning their cytotoxic potential with great success in vitro, ex vivo in primary leukemia cells from children with primary and relapsed ALL and in vivo in a lymphoma model in mice. We could show anti-leukemic and anti-lymphoma activity in various established cell lines, like Nalm6, Reh, SupB15 and BJAB. By Casy Counter we detected a decrease of proliferation and vitality in cell culture. That the cell death is effected by apotosis induction we investigated by flowcytometric measurement of DNA-fragmentation after propidiumiodid staining (LC50 = 5–10 μM) and phosphatidylserin-staining on cell membrane surface. Via western blotting we could show titanocene-induced caspase-processing and PARP cleavage. To characterize the titanocene apoptosis pathway we used cellular model systems with deficiencies in different molecular parts of the apoptosis cascade. The induced apoptosis reveals to be independent of CD95 receptor, the pro-apoptotic factor SMAC and the anti-apoptotic protein Bcl-2. Even in mamma carcinoma cells (MCF-7) with Caspase 3- deficiency titanocenes were able to induce apoptosis significantly. We also investigated a loss of mitochondrial membrane potential, which indicates the involvement of the mitochondrial apoptosis machinery in titanocene-induced cell death. Unspecific cytotoxic effects, like necrosis, could be excluded by lactatdehydrogenase-release assay. Ex vivo we tested isolated primary lymphoblasts of children with primary and relapsed ALL. We could demonstrate, that even lymphoblasts with resistance against doxorubicin (p < 0.005) and cytarabine (p < 0.01) are sensitive to titanocenes. In vivo tests in a mouse model with human lymphoma cells (BJAB) show significant reduce of tumor progression effected by oral treatment with carbonyl-substituted titanocenes compared with a control group. These experiments in mice show a good tolerability of titanocenes. Taken together, carbonyl-substituted titanocenes comprise a totally new, very promising class of cytostatic agents for lymphoma and leukemia therapy, especially for the therapy of relapsed ALL in childhood.
Clinical Significance of PML/RAR Isoform in Patients of Acute Promyelocytic Leukemia.
Authors: Kim Ki-Hyang , Lee Won-Sik , Lee Kyoo-Hyung , Lee Je-Hwan , Choi Seong-Jun , Lee Jung-Hee , Park Jae-Hoo , Min Young-Joo , Kim Hawk , Bae Sung-Hwa , Ryoo Hun-Mo , Hyun Myung-Soo , Kim Min-Kyung , Zang Dae-Young , Kim Hyo-Jung , Ahn Jin-Seok , Jung Chul-Won , Lee Kyeong-Won , Lee Jung-Lim , Joo Young-Don
Published: October 2019

Background: Three types of PML-RARα mRNA fusion transcripts in acute promyelocytic leukemia (APL) could be existed: a short (S)-form type, a long (L)-form type or a variable (V)-form type. Whether 3 types of PML-RARα mRNA fusion transcripts associated with different manifestations and outcomes in individual APL cases are unclear. Recently, some studies reported the controversial results for the relationship between the types of PML-RARα mRNA fusion transcripts and clinical outcomes. But, there was no data about the types of PML-RARα mRNA fusion transcripts especially for the APL patients who were received remission induction therapy with AIDA.

Methods: We performed a retrospective analysis for the data of 94 patients with APL, whose isoform data was available. We evaluated the differences of therapeutic outcome of remission induction chemotherapy in terms of response rate, relapse-free survival (RFS), overall survival (OS) and the association of pretreatment clinical parameter characteristics according to the PML-RARα isoforms.

Results: The median age of the patients was 41 years (15–85). CR rate following remission induction treatment was 84.9% (AIDA 87.0% vs. non-AIDA 80.0%). Among 94 patients, there were 58 L-form cases (62.1%), 32 S-form cases (34.0%), 4 V-form cases (4.3%). There was no significant difference at any patient’s pretreatment characteristic according to PML-RARα isoform type. CR rate was higher in the group of initial WBC <10,000/ul (93.5% vs. 65.4%, p=0.001). But there was no difference within the isoform L/S subgroup (84.2% vs. 87.2%). And OS and RFS were not different between isoform L/S subgroup (5yr 74.3% vs. 83.1%, 84.2% vs. 85.1%). AIDA induction group was better than non-AIDA induction group regarding OS and RFS (5yr 84.4% vs 55.7%, p=0.026, 90.0% vs 65.7%, p=0.007), but not significant in the multivariate analysis. And also, it was not significantly different in the OS and RFS between isoform L/S subgroup of the AIDA induction group (5yr 80.5% vs. 92.0%, 95.7% vs. 97.0%).

Conclusion: This study suggests that

high initial WBC count is associated with low CR rate, AIDA induction group has a trend of better OS and RFS, treatment outcomes according to PML-RARα isoform type are not different.

Prospective study will be needed to confirm the meaningful significance of PML-RARα isoform type.
Prognostic Significance of Molecular-Cytogenetic Abnormalities in Pediatric T-ALL Is Not Explained by Immunophenotypic Differences.
Authors: van Grotel Martine , Langerak Anton W. , Beverloo H. Berna , Buijs-Gladdines Jessica G.C.A.M. , Burger Nicole B. , van Noesel Max M. , Pieters Rob , Meijerink Jules P.P.
Published: October 2019

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by recurrent chromosomal rearrangements that may have prognostic significance. T-ALL cases with specific aberrations may arrest at specific T-cell development stages. Two classification systems were developed to classify T-ALL into developmental subgroups, i.e. the European Group for the Immunological Characterization of Leukemias (EGIL) and the T-cell receptor (TCR) classification system. In this study, we investigated the relationship between molecular-cytogenetic abnormalities and T-cell development stage. We investigated whether arrest at specific T-cell stages explains the prognostic significance of molecular-cytogenetic abnormalities. To this aim, we extensively studied 72 pediatric T-ALL cases by FISH and RQ-PCR for the presence of genetic abnormalities and expression of transcription factors, by PCR and sequencing for NOTCH1 mutations and by flow-cytometry to determine the T-cell receptor status as well as CD marker expression. The median clinical follow-up was 5 years. HOX11 rearranged cases were CD1 positive consistent with a cortical stage, but as 4 out of 5 cases lacked cytoplasmatic-beta expression, developmental arrest may precede beta-selection. HOX11L2 was especially confined to immature and pre-AB developmental stages, but 3 out of 17 HOX11L2 mature cases were restricted to the γδ-lineage. TAL1 rearrangements were restricted to the αβ-lineage with most cases being TCRαβ positive. NOTCH1 mutations were present in all molecular-cytogenetic subgroups without restriction to a specific developmental stage. The CALM-AF10 translocation as detected in 3 T-ALL patients was significantly associated with early relapse. TAL1 or HOX11L2 rearrangements were associated with trends to good and poor outcome, respectively. Cases with high TAL1 expression levels also demonstrated a trend towards good outcome, whereas cases with the lowest TAL1 levels had a poor outcome and were mostly HOX11L2 or CALM-AF10 positive. NOTCH1 mutations did not predict for poor outcome. Classification into T-cell developmental subgroups did not predict for outcome. In conclusion, the present study shows that differences in outcome for various molecular-cytogenetic subgroups cannot be attributed to differences in T-cell maturation stage.
Myeloid-Associated Antigen Expression Is an Adverse Factor for Complete Remission Following Induction Chemotherapy of Adult Precursor T-Lymphoblastic Leukemia/Lymphoma (T-ALL).
Authors: Al-Khabori Murtadha , Samiee Samira , Fung Sharon , Xu Wei , Brandwein Joseph , Brien William , Patterson Bruce , Chang Hong
Published: October 2019

We retrospectively reviewed our experience of 63 adult T-ALL patients to identify clinical and pathologic prognostic factors and build a risk-stratification model for induction chemotherapy. At presentation, the patients’ median age was 30, 49 were male and 14 female, 69% had lymphadenopathy, 38% a mediastinal mass, 24% CNS involvement and 21% splenomegaly. The median initial WBC was 17.9 x 109/L (range 0.10–510.0). Blasts expressed CD34 in 42% of cases, CD10 in 33% and at least one myeloid-associated antigen (CD13 or CD33) in 27%. Karyotypes were abnormal in 34% of cases. Fifty-three of 61 patients (87%) who underwent induction chemotherapy achieved complete remission (CR) on protocols including vinca alkaloids, anthracyclines and corticosteroids. On univariate analysis; age, gender, initial WBC, CD10, CD34 and abnormal karyotype did not predict CR but patients expressing at least one myeloid-associated antigen had a CR of 71% compared to 93% (P=0.03) for patients not expressing myeloid antigens. The median follow-up was 19.2 months (95% CI: 0.1–172.8). Twenty-three patients relapsed with a median relapse-free survival (RFS) of 41.6 months (95%CI: 21.6–55.9). The RFS was longer for patients with an initial WBC of 3–50 x 109 and for cases expressing CD10 but neither was statistically significant (P=0.19, P=0.14). On follow-up, 34 patients died with a median overall survival (OS) of 31.3 months (95%CI: 16.8–56.8). Patients with CD10 expression had a longer median OS at 44.7 months (versus 19.2 months for CD10 negative patients) but again, the difference was not statistically significant (P=0.11). Age, gender, CD34, myeloid-associated antigen expression and karyotype did not influence RFS or OS. Our study indicates that expression of myeloid-associated antigens is an adverse prognostic factor for complete remission of adult T-ALL and should be considered for induction chemotherapy risk-stratification.
Crosslineage T-Cell Receptor Genes Rearrangements and Active Receptor Editing in B-Cell Acute Lymphoblastic Leukemia.
Authors: Makuch-Lasica Hanna , Majewski Miroslaw , Nowak Grazyna , Kania Iwona , Lewandowska Monika , Konopka Lech , Warzocha Krzysztof
Published: October 2019

B-cell acute lymphoblastic leukemia (B-ALL) results from clonal expansion of B-lymphocytes derived at different stage of differentiation. Immunoglobulin (Ig) heavy chain genes (IGH), light chain kappa (IGK) and lambda (IGL) genes rearrange during early B-lymphocyte differentiation. T-cell receptor (TCR) genes are considered to rearrange exclusively in normal T lymphocytes, but malignant B lymphoblasts often contain crosslineage rearranged TCR genes. The clonal leukemic cell population, carrying identical copies of rearranged Ig and/or TCR genes, can be identified above 95% of B-ALL patients. In our study Ig/TCR genes rearrangements were detected by multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol. DNA was isolated by column method from mononuclear cells isolated from the peripheral blood/bone marrow samples obtained at initial diagnosis from 36 B-ALL patients. Monoclonal rearrangements of Ig genes were detected in 100% (36/36) of patients. The most frequent rearrangements were observed in IGH genes (94%), including complete IGHV-IGHJ in 83% (30/36) and incomplete IGHD-IGHJ in 22% (8/36) of patients. Among complete IGH rearrangements 2 biallelic rearrangements in IGHV1-7 and IGHJ genes (FR3) were found. Ig light chain genes rearrangements were identified in 26 patients (72%) (including 64% of IGKV-IGKJ, 47% IGKV/intron-Kde, and 22% IGLV-IGLJ) what indicates active receptor editing occurring during B lymphoblasts leukemogenesis. Crosslineage TCR genes rearrangements were found in 97% (35/36) of patients. TCR beta genes rearrangements were detected in 47% (17/36) of patients (complete TRBV-TRBJ in 25% (9/36), TRBD-TRBJ in 6/36 patients - 17%). TRGV-TRGV in 58% (21/36), TRDV-TRDJ in 58% (21/36); 17 monoallelic and 4 biallelic were found. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL genes rearrangements incidence, present allelic exclusion and active receptor editing in B-ALL patients. B-ALL lymphoblast undergoes rearrangement on the same IGK allele before IGL genes rearrangement occur. The data may suggest the possible of antigens in B-ALL immunopathogenesis. The results indicate also rearranged IGK, IGL and TCR genes as stable molecular marker for monitoring MRD in B-ALL.
Clinical Characteristics and Outcomes in Patients with t(8;21) Acute Myeloid Leukemia in Japan.
Authors: Narimatsu Hiroto , Yokozawa Toshiya , Iida Hiroatsu , Tsuzuki Motohiro , Hayakawa Masaya , Takeo Takaaki , Iino Masaki , Ichihashi Takuji , Kato Chiaki , Sawamoto Akiyo , Sao Hiroshi , Yanada Masamitsu , Emi Nobuhiko , Kiyoi Hitoshi , Yamaguchi Tetsushi , Naoe Tomoki , Suzuki Ritsuro , Sugiura Isamu
Published: October 2019

Clinical characteristics of Japanese patients with t(8;21) acute myeloid leukemia (AML) have not been well described. From January 2000 to December 2005, a total of 147 Japanese adult de novo AML (FAB: M2) patients were newly diagnosed with t(8;21) AML (n=46) or without t(8;21) AML (n=101) in collaborating hospitals. Patients with t(8;21) (median age, 49.5 years; range, 18–86 years) were significantly younger than AML(M2) patients without t(8;21) (median age, 60 years; range 17–90 years) (p<0.001). Three-year overall survival rate in patients with t(8;21) was 70% (95% confidence interval (CI), 51–83%), significantly better than that in AML (M2) patients without t(8;21) (log-rank test, p=0.005) (Figure). Among patients <60-years-old, overall survival rates of patients with t(8;21) AML and patients with non-t(8;21) AML(M2) were 71% (95%CI, 47–86%) and 58% (95%CI, 41–72%), respectively (log-rank test, p=0.28). Of the 40 patients who achieved complete remission, 21 patients received high-dose cytarabine-containing consolidation therapy. Event-free survival rates at 3 years after diagnosis in patients with and without high-dose cytarabine were 60% (95%CI, 36–78%) and 57% (95%CI, 26–79%), respectively (log-rank, p=0.87). In multivariate analysis, age and white blood cell count at diagnosis represented significant predictors of overall survival. For the 147 AML(M2) patients, presence of t(8;21) was not a significant predictor of overall survival after adjusting for age (hazard ratio, 0.65; 95%CI, 0.34–1.24; p=0.19). Japanese patients with t(8;21) AML display more favorable survival rates than those in Western countries. Efficacy of high-dose cytarabine might differ between Japanese and Western patients. Clinicians must be aware of potential differences among different ethnicities.

Figure Figure
Immunohistochemical Staining for p16 in Acute Lymphoblastic Leukemia.
Authors: Kim Sun Young , Jeong Dae-Chul , Lee Mee Jeong
Published: October 2019

Purpose: p16 gene, mapped to the 9p21 chromosomal region, has emerged as a candidate tumor suppressor gene in human neoplasm. It is an inhibitor of cyclin-dependent kinase and inhibits Rb phosphorylation. In a variety of tumors, deletion and/or mutation of the p16 gene has been found including childhood acute lymphoblastic leukemia (ALL). Despite their high frequency, the prognostic importance of p16 alterations is still controversial in ALL and has been reported to be either unfavorable or similar to that of other patients. We studied the correlation between p16 and clinical outcomes of patients diagnosed as ALL.

Methods: We performed an immunohistochemical staining for protein p16 in 74 cases of bone marrow biopsy slide initially diagnosed as ALL between January 1998 and December 2006. We reviewed the clinical manifestations, laboratory findings, treatment outcomes retrospectively.

Results: Of 74 slides, 12 were negative for p16. Seven were males and 5 were females with a median age at diagnosis was 5.8(1.3–18.8) years. Initial WBC were 17,225(500–403,300)/μl and by immunologic surface marker analysis, 7 patients were early pre-B CALLA(+) and 5 patients were T-cell ALL. Two patients of intermediate risk group had relapsed and died. Three patients had family history of breast cancer. Four patients died and overall survival rates were 53.5±18.7%.

Conclusion: p16 is supposed to be an independent risk factor of childhood ALL associated with poor outcomes. In clinical setting, the clinician must take into account p16 status, not only at the genomic but also at the protein level. Further clinical experience on thoroughly investigated cases will help a better understanding between p16 and clinical outcomes.
The MDM2 Antagonist Nutlin-3 Is a Potent Inducer of Apoptosis in Pediatric Acute Lymphoblastic Leukemia Cells with Wild-Type p53 and Overexpression of MDM2.
Authors: Gu Lubing , Zhu Ningxi , Findley Harry W. , Zhou Muxiang
Published: October 2019

In pediatric acute lymphoblastic leukemia (ALL), overexpression of MDM2 by leukemic cells is typically associated with a wild-type (wt) p53 phenotype and chemoresistance. A recently-developed small-molecule antagonist of MDM2, nutlin-3, inhibits the MDM2-p53 interaction, resulting in induction of p53 activity and apoptosis. In the present study, we evaluated the cytotoxic effect of nutlin-3 on ALL cells with different p53 status and MDM2 expression, using 18 cell lines and 30 primary leukemia samples. We found that both ALL cell lines and primary ALL samples with wt-p53 are sensitive to nutlin-3. No cytotoxic effect of nutlin-3 was detected in ALL cells with either p53-mutant or null phenotypes. In wt-p53 ALL cells, there was a significant positive correlation between MDM2 expression levels and sensitivity to nutlin-3. Nutlin-3-induced cell death was mediated by p53-induced activation of pro-apoptotic proteins and by p53-induced repression of the anti-apoptotic protein survivin. Because p53 function is inhibited by MDM2 in chemoresistant, MDM2-overexpressing ALL cells, potent killing of these cells by nutlin-3 suggests that this agent may be a novel therapeutic for refractory ALL.
Sensitive NOD-scid-IL2γnull (NOG-scid) Assay for Human Precursor B Acute Lymphoblastic Leukemias (ALL).
Authors: Morisot Sebastien , Hildreth Richard , Kaplan Ian M. , Stetler-Stevenson Maryalice , Taylor Julie A. , Ahuja Yachna , Bloom Lauren , Wayne Alan S. , Civin Curt I.
Published: October 2019

Results of treatment for childhood ALL have improved considerably over the past few decades, yet still ∼20% of pediatric patients relapse. New in vivo models of human ALL might increase understanding of ALL biology and speed preclinical development of novel treatment regimens. Multiple strains of immunodeficient mice support growth of human ALL cells (Bachmann, Current Drugs Target 2007, review). The recently described highly immunodeficient NOD-scid-IL2γnull (NOG-scid) mouse strain offers a number of advantages over the widely-used NOD-scid strain: NOG-scid mice have a normal lifespan, do not develop spontaneous lymphomas, and require ∼10-fold fewer normal human hematopoietic stem-progenitor cells to generate human hematopoiesis (Hiramatsu, Blood 2003; Ishikawa, Blood 2005). In addition, NOG-scid mice support generation of human leukemia from transplanted human acute myeloid leukemia (AML) (Ninomiya, Leukemia 2007). Herein we report a sensitive in vivo model using transplant of human childhood precursor B ALL cells into sublethally irradiated NOG-scid mice. Transplanted mice developed fatal leukemia 4–5 weeks after intravenous injection of 5x104 REH or KOPN8 cells (established precursor B ALL cell lines derived from pediatric ALL patients). At necropsy, the mice had massive splenomegaly and hyperplastic bone marrow. Blood, spleen, and marrow contained abundant human cells with immunophenotype matching the transplanted ALL cell line. We went on to similarly transplant primary leukemic blast cells from pediatric ALL patients. In 3 of 5 tested primary ALL cases, fatal human leukemias developed in the NOG-scid mice. In titrations of 103-2.5x106 transplanted ALL cells, time to clinical leukemia was dose-dependent, and 1000 primary human cells was sufficient to initiate leukemia development. In addition, we detected human leukemic blast cells by flow cytometric immunophenotyping in the blood of transplanted NOG-scid mice as early as 10 days post-transplant, enabling objective leukemia diagnosis ∼4 weeks prior to clinical signs.

CONCLUSIONS: NOG-scid mice supported the generation of human precursor B ALL from cell lines and primary ALL cases, objectively detectable as early as 10 days post-transplant. Our dose titration results suggest that this model detects a high frequency of ALL-initiating cells. This in vivo model may provide a powerful assay both for fundamental questions regarding the biology of leukemia stem cells and for preclinical studies of novel anti-neoplastic agents and regimens.
Frequent Occurrence of Aberrant Pax5 Splice Variants in Philadelphia Chromosome-Positive Lymphoblastic Leukemia.
Authors: Okawara Akemi , Tojo Arinobu
Published: October 2019

Pax5 is a key regulator of B cell commitment and is indispensable throughout the early stages of B cell differentiation. B cell development is blocked at the pro-B cell stage in Pax5-null mice. On the other hand, several investigators have reported that aberrant Pax5 transcripts as well as forced expression of Pax5 result from genetic and/or epigenetic mechanisms especially in B cell malignancies, suggesting the involvement of Pax5 abnormality in development and progression of B cell malignancies. In the present study, we examined Pax5 expression in Philadelphia chromosome (Ph)-positive lymphoid malignancies including 17 cases of Ph-positive acute lymphoblastic leukemia (Ph-ALL) and 3 cases of lymphoid blast crisis of chronic myeloid leukemia (CML-BC). By the reverse transcription-polymerase chain reaction (RT-PCR) method, we detected 4 types of Pax5 isoforms among those. In 16 samples, full-length as well as truncated forms of Pax5 were expressed. Expression of Pax5 could not be detected in 3 cases, and was only faintly found in the remaining one. These 4 cases did not express or very weakly expressed EBF, early B cell transcription factor upstream of Pax5. Sequencing analysis revealed that all 4 truncated isoforms were derived from exon skipping as follows. Type I is lacking in exon 2 and exon 3 (D2/3), type II shows deletion of exons 2–6 (D2-6), type III; D2-7, and type IV; D2-8. Two isoforms resulted in frame-shift, and two isoforms were in-frame splice variants without exons coding DNA binding domain. Transactivation potential of these two in-frame variants was examined by transfection of 293T cells with a reporter plasmid containing high-affinity Pax5 binding sites from the CD19 gene linked to the minimal TATA-like promoter, indicating much lower transcriptional activity in comparison to wild-type Pax5. These results suggest the frequent occurrence in adult Ph-positive lymphoid malignancies of aberrant Pax5 splice variants with reduced transcriptional activity, leading to impairment of normal B cell differentiation.
Analysis of the Role of Wnt Signaling for the Interaction between Leukemia Cells and Stroma.
Authors: Abe Akihiro , Niwa Yosuke , Xu Jinglan , Suzuki Momoko , Hayakawa Fumihiko , Katsumi Akira , Kiyoi Hitoshi , Naoe Tomoki
Published: October 2019

Wnt signaling plays an important role in hematopoietic stem cell self-renewal and proliferation. Recent results also revealed that an aberrant activation of Wnt signaling has been related with hematological malignancies. We have previously reported a stroma-dependent human leukemia cell line, TRL-01, which could not be maintained by any cytokines without stroma or on fibronectin-coated plate although they periodically stimulated the proliferation. We here analyzed the role of Wnt signaling on the cell-adhesion, proliferation and survival of this cell line. TRL-01 was maintained on human bone marrow stroma cell line, hTERT. The apoptosis of TRL-01 on hTERT was suppressed by Wnt-receptor competitor, secreted Frizzled related protein (sFRP)-1, or Rho kinase inhibitor, Y29632 but not by specific inhibitor of canonical pathway, DKK-1. However, the apoptosis of TRL-01 without hTERT was not affected by sFRP-1, Y29632 or DKK-1. These results suggest that survival of TRL-01 on stroma was regulated by non-canonical pathway of Wnt signaling. Next, the effects of Wnt3A, 5A and 10B on TRL-01 were analyzed by establishing hTERT transduced with Wnt3A, 5A or 10B genes. Wnt3A is known as an important factor for the stimulation of self-renewal and proliferation of hematopoietic stem cells, and Wnt5A and 10B were reported to be expressed in B-ALL. When TRL-01 was cocultured with hTERT/Wnt3A or 10B, adhesion was reduced and β-catenin was significantly increased. The condition medium from hTERT/Wnt3A or 10B also stimulates the accumulation of β-catenin but inhibited the survival and proliferation of TRL-01. Wnt5A did not have any significant effects. These results suggest that Wnt3A and 10B stimulate canonical pathway with accumulation of β-catenin but negatively regulate the survival and proliferation of TRL-01. Comparing the expression pattern of Wnt transcripts of TRL-01 and hTERT by RT-PCR, Wnt5A, 5B and 14 were found to be dominantly expressed in hTERT. The role of Wnt5B and 14 for the interaction between TRL-01 and hTERT remains to be elucidated.
In Vitro Study of the Effect of Cyclosporine A, Tetrandrine and Their Combination on the Reversion of Multidrug Resistance in Leukemia Cell Line.
Authors: Chen Bao-An , Guo Jing-jing , Gao Feng , Gao Chong , Chen Jian , Sun Yun-Yu , Wang Jun , Zhao Gang , Ding Jia-Hua , Chen Ning-Na , Pei Xiao-Ping , Dai Yong-Yuan , Shun Qian
Published: October 2019

Objective: The aim of this study was to investigate the mechanism to reverse the drug resistance of leukemia cells in Cyclosporine A(CsA), tetrandrine (Tet) alone or their combination by using empirical methods, and to provide the basic evidence for further study, and lay the theoretical basis for the clinical applications.

Method: By MTT assay, the IC50(the concentration causing 50% inhibitor of cell growth) of DNR was determined;By flow cytomet ry (FCM) assay, the intracellular DNR concentration and the expression of P-glyco-protein (P-gp) were examined, and the apoptotic changes were observed by Anexin-V. By fluorescent semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), the mRNA expression of multidrug resistance (MDR) was measured.

Results:

The IC50 of DNR for K562 and K562/A02 cells were 0.26mg/l and 21.22mg/l respectively. Treating K562/A02 cells with CsA(1mg/L), TTD(0.1mg/L)and both for 48 hours partially restored the sensitivity of K562/A02 cells to DNR(IC50 were 7.83 mg/L,5.32 mg/L,2.33 mg/L respectively.) but had not effect on K562 cells; K562/A02 cells showed apoptotic characteristics after treated with CsA, and both for 48 hours(Apoptosis rate was 10.27%,17.64%,52.79% respectively); The fluorescence intensity of intracellelar DNR in K562 and K562/A02 cells treated with DNR(1mg/L)was 522,173 respectively. CsA and TTD (alone or combination) elevated the intracellular DNR concentration in K562/A02 cells(the fluorescence intensity of intracellelar DNR in K562/A02 cells was 188,339,515 respectively); The fluorescence intensity of P-gp in K562 and K562/A02 cells was 0.5,97.97. The P-gp expression was down after treated with CsA, TTD and both(73.7,65.05,58.1); mdr1 mRNA was also down regulated, and the effect of their combination was greater. Date was analyzed by SPSS 13.0 software and expressed as mean ± SD.

Conclusions:

High expression of P-gp may be involved in the mechanism of multidrug resistance (MDR) of K562/A02 cells line; MDR can be partially reversed by CsA or TTD, of which the combination shows a great synergistic reversal effect.
The Reduction of Leukemic Blasts in Bone Marrow Aspirate on Day 6 of Remission Induction Treatment Is Predictive for Complete Remission Rate and Survival in Adult Acute Myeloid Leukemia; the Results of Multicenter, Prospective Polish Adult Leukemia Group Trial.
Authors: Holowiecki Jerzy , Grosicki Sebastian , Kyrcz-Krzemien Slawomira , Kuliczkowski Kazimierz , Kielbinski Marek , Skotnicki Aleksander B. , Jakubas Beata , Hellmann Andrzej , Robak Tadeusz , Seweryn Marek J. , Stella-Holowiecka Beata , Sadus-Wojciechowska Maria
Published: October 2019

The goal of this study was to prove the significance of an early evaluation of leukemic blast reduction based on cytological examination of bone marrow aspirates obtained on day 6 of remission induction treatment. Patients. 90 adult AML patients aged 18–60 (median 47) registered to the PALG prospective trial evaluating the efficacy of 3 remission induction protocols:

DAF: daunorubicine (DNR) 60 mg/m2/d iv, d 1–3; cytarabine (AraC) 200 mg/m2/d ci, d 1–7, and fludarabine 25 mg/m2 2h inf. iv d 1–5), DAC (like DAF but cladribin is used at 5 mg/m2 instead of fludarabine) and the standard DA 3+7 regimen.

Bone marrow aspirates were performed before the treatment and on the 6-th day of the first remission induction course, and the MGG stained marrow smears were evaluated at each centre by two experienced hematologists.

Results. Based on the proportion of blasts in bone marrow specimen on day 6 patients were arranged into 2 groups:

“responders”with ≤5% of blasts (n=59) and “non responders” with >5% of myeloblasts in bone marrow (n=31).

The complete remission rate (CR) in the first group equalling 89% (51/59) was significantly higher in comparison to that obtained in the second group 29%(9/31), p=0,008. The probability of overall survival (OS) was also higher in the group of “responders” if compared to “non responders”; 65% versus 45% respectively, p=0,009. In multivariate analysis including bone marrow examination, cytogenetic risk group, age, leukocyte count at diagnosis and the induction arm only the persistence of leukemic blasts >5% in bone marrow specimen on 6-th day of induction was associated with higher risk of not achieving CR (HR = 52,6; p=0,00002), and with shorter OS (HR=3,13; p=0,02). Conclusion. This prospective, multicenter study demonstrates that a simple and commonly accessible evaluation of the leukemic blasts reduction in bone marrow smear on the 6-th day of remission induction treatment is a reliable and independent predictor for achieving CR and for overall survival. This offers a decision point for an early modification of the treatment strategy.
Partial Efficacy of Bortezomib on Acute Myeloid Leukemia (AML) Cells in a Patient with Concomitant Multiple Myeloma and AML.
Authors: Joly Bertrand , Quillet Patrick , Salanoubat Celia , Bouledroua Selwa , Petitdidier Charlotte , Devidas Alain
Published: October 2019

Bortezomib has a well known efficacy in multiple myeloma, but effects on AML cells is only observed in vitro and remain to be clinically proved. In a 78 year-old patient, we diagnosed two concomitant aggressive hematological malignancies: IgA Kappa multiple myeloma and de novo FAB-M5 AML. The patient was consulting for bone pain and progressive bicytopenia. The bone marrow was infiltrated with 30% dystrophic plasmocytes and 35% myeloïd monoblastes with CD13+, CD33+ phenotype. A serum M component of 5 g/dl IgA kappa was present at diagnosis with mild acute renal failure, elevated beta2microglobulinemia and IPSS score of 2. The cytogenetic study revealed poor prognosis factors for both malignancies with a complex caryotype including chromosome 13 abnormalities and MLL rearrangement. Standard treatments to cure both diseases one after the other could not be a good option, due to the presence of these poor prognosis factors. Thus, we decided to treat the patient with Bortezomib, a proteasome inhibitor known to induce clinical response in multiple myeloma and apoptosis of leukemia cell lines in vitro. After two cycles of Bortezomib (1.3 mg/m2 day 1,4,8,11) combined with steroids (dexamethasone, 40 mg day 1–4), the patient obtained a near complete myeloma remission with normal electrophoresis, but also disappearance of circulating AML blast and a 70% reduction of medullary AML blasts. Low-dose cytarabine (15 mg twice the day for 10 days) was introduced after the fourth cycle of bortezomib because of persistent circulating AML blasts. Bortezomib was stopped after 5 cycles because of neurological adverse event. After 5 cycles of cytarabine, the patient reached complete cytogenetic remission of both diseases. The patient still remains in complete remission after 13 months of follow-up. We conclude that bortezomib had a partial efficacy on AML cells in vivo and could have synergized with cytarabine to reach and maintain complete remission of both poor prognosis AML and multiple in this patient.
CD8+CD28− in Peripheral Blood of Patients with Cutaneus T-Cell Lymphoma (CTLC) in Different Clinical Stages.
Authors: Urbaniak-Kujda Donata M. , Kapelko-Slowik Katarzyna L. , Jazwiec Bozena , Maj Joanna , Dybko Jaroslaw , Slowik Miroslaw , Wolowiec Dariusz , Frydecka Irena , Kuliczkowski Kazimierz
Published: October 2019

Background: Patients with CTLC usually have a poor immune response. CD8+CD28− T cells are new types of immune suppressor cells. The study was to analyze the level and changes of them in peripheral blood with CTLC, their effects on immunosupression of CTLC, the influence factors to provide reference for treatment of CTLC patients.

Patients and Methods: 27 patients with CTLC were enrolled in the study: 8 women and 19 men aged 35–81, medium 57. According to Ann Arbor classification stage I was represented by 1 patient, stage II -6 patients, stage III - 5 patients, stage IV - 6 patients and stage early stage (premycoticus) was represented by 8 patients. Peripheral blood samples were assessed from all patients with CTLC before treatment and 8 normals. Percentage CD8+CD28− T cells was analyzed by flow cytometry.

Result: Compared with control group percentage of CD8+CD28− T cells was significantly higher in patients group p=0,005 (test U Mann Whitney). There were no significant differences of T-cells in among patients with different clinical stages. We revealed the correlation between CD8+CD28− cells and advanced clinical stage r=0,54, p=0,035 (Spearman correlation test).

Conclusion: Percentage of CD8+CD28− T cells is increased in peripheral blood in CTLC patients, and correlates with advanced stage.
Response of Hodgkin Lymphoma Patients of Different Ethnic Groups to Standard ABVD with or without Involved Field RT.
Authors: Ofori-Ntow Samuel , Ciobanu Bety , Telfer Margaret Clare , Nathan Sunita , Kovarik Paula , Kouz Rafid , Griza Decebal S. , Lad Thomas , Catchatourian R.
Published: October 2019

Background: The phenotypic expression of Hodgkin lymphoma among different ethnic groups is not well described. We conducted a study to look at the characteristics and the clinical response of Hodgkin lymphoma patients of different ethnicities to standard treatment.

Method: A retrospective analysis of 65 Hodgkin lymphoma patients diagnosed 2003 through 2006 was undertaken. Patients who have been compliant and completed assigned chemotherapy (ABVD) with or without Involved Field RT were evaluated for response to treatment using CT scan and PET scan. Data was analyzed using fisher exact test.

Results: Patients with limited staged disease received 4 to 6 cycles of ABVD +/− RT while those with advanced staged disease received 6 to 8 cycles of ABVD +/− RT. All 50 patients achieved objective response. Complete response rates were 52% Hispanic, 68% African American, and 75% Caucasians. (p=0.4)

Conclusion: Hodgkin lymphoma is as common in Hispanics as in African American. The mixed cellularity subtype is more common in the Hispanics; however the nodular sclerosis subtype remains the most common histological subtype. Though more Hispanics had an early clinical stage disease at diagnosis, they had a lower complete response rate compared to the other ethnic groups. How this observation will translate into a poorer survival outcome remains to be seen.

Results: Hispanic African American Caucasians N=65 26 27 12 Median age 26 27 38 Nodular Sclerosis subtype 26 27 12 (P=0.08) Mixed Cellularity subtype 6 1 1 Clinical Stage 1–2 16 9 9 (P=0.03) Clinical Stage 3–4 10 18 3 Completed Treatment N=50 23 19 8