[Clinical significance of epidermal growth factor receptor and thymidylate synthase expression in primary liver cancer].

Objective: To investigate epidermal growth factor receptor (EGFR) and thymidylate synthase (TS) expression in primary liver cancer, and analyze its clinicopathological features and prognostic significance. Methods: Immunohistochemistry was performed using EnVision method to detect EGFR and TS expression in 41 cases of liver cancer. Correlation coefficient between EGFR and TS was calculated by Spearman method. Fisher's exact probability method or χ(2) test was used to analyze the clinicopathological features of EGFR and TS. Kaplan-Meier method was used to calculate the survival rate of patients in conjunction with the log-rank test.COX proportional hazard regression model was used to analyze the prognostic factors of patients. ROC curve was used to analyze the predictive accuracy of EGFR and TS for prognosis. Results: The positive rates of EGFR and TS in liver cancer tissues were 34.15% and 39.02%, respectively. There was a positive correlation between EGFR and TS expressions, and the difference was statistically significant (P < 0.05). EGFR was associated with tumor size and tissue differentiation (P < 0.05) in HCC patients, whereas TS was associated with tissue differentiation (P < 0.05). There was no significant difference in prognostic effect of EGFR on survival rate (P > 0.05). TS prognostic effect on survival rate was statistically significant (P < 0.05). HR of EGFR was 0.210 with 95% CI, 0.052-0.852, P = 0.029; indicating that the risk of death in patients with negative EGFR was 0.210 times higher than that in patients with positive EGFR. HR of TS was 2.496, with 95% CI, 1.325-4.701, P = 0.005, indicating that the risk of death increased by 2.496 times with the same level of EGFR. The area under the EGFR curve was 0.553 and its approximate reference confidence interval was 95% (0.355, 0.751), indicating that EGFR was a risk factor for death and the area under the TS curve was 0.695, and its approximate reference confidence interval was 95% (0.513, 0.878), indicating that TS was a risk factor for death. Conclusion: EGFR and TS were equally expressed in primary liver cancer, and EGFR and TS expressions were positively correlated. EGFR and TS had an effect on the degree of tissue differentiation in patients with liver cancer. EGFR and TS were risk factors for prognosis, and TS may assist EGFR.

Quantitative phase-field lattice-Boltzmann study of lamellar eutectic growth under natural convection.

The influence of natural convection on lamellar eutectic growth was determined by a comprehensive phase-field lattice-Boltzmann study for Al-Cu and CBr_{4}-C_{2}Cl_{6} eutectic alloys. The mass differences resulting from concentration differences led to the fluid flow and a robust parallel and adaptive mesh refinement algorithm was employed to improve the computational efficiency. By means of carefully designed "numerical experiments", the eutectic growth under natural convection was explored and a simple analytical model was proposed to predict the adjustment of the lamellar spacing. Furthermore, by alternating the solute expansion coefficient, initial lamellar spacing, and undercooling, the microstructure evolution was presented and compared with the classical eutectic growth theory. Results showed that both interfacial solute distribution and average curvature were affected by the natural convection, the effect of which could be further quantified by adding a constant into the growth rule proposed by Jackson and Hunt [Jackson and Hunt, Trans. Metall. Soc. AIME 236, 1129 (1966)].

Morphology transition of the primary silicon particles in a hypereutectic A390 alloy in high pressure die casting.

The microstructure of a high-pressure die-cast hypereutectic A390 alloy, including PSPs, pores, α-Al grains and Cu-rich phases, was characterized using synchrotron X-ray tomography, together with SEM, TEM and EBSD. The Cu-rich phases exhibited a net morphology and distributed at the boundaries of the α-Al grains, which in turn surrounded the PSPs. Statistical analysis of the reconstructed 1000 PSPs showed that both equivalent diameter and shape factor of the PSPs exhibited a unimodal distribution with peaks corresponding to 25 µm and 0.78, respectively.) PSPs morphology with multiple twinning were observed and morphological or growth transition of the PSPs from regular octahedral shape (with a shape factor of 0.85 was mainly caused by the constraint of the Cu-rich phases. In particular, the presence of the Cu-rich phases restricted the growth of the α-Al grains, inducing stress on the internal silicon particles, which caused multiple twinning occurrence with higher growth potential and consequently led to growth transitions of the PSPs.

Newly diagnosed acute lymphoblastic leukemia in China (I): abnormal genetic patterns in 1346 childhood and adult cases and their comparison with the reports from Western countries.

It has been generally acknowledged that the diagnosis, treatment and prognosis evaluation of leukemia largely rely on an adequate identification of genetic abnormalities. A systemic analysis of genetic aberrations was performed in a cohort of 1346 patients with newly diagnosed acute lymphoblastic leukemia (ALL) in China. The pediatric patients had higher incidence of hyperdiploidy and t(12;21) (p13;q22)/ETV6-RUNX1 than adults (P<0.0001); in contrast, the occurrence of Ph and Ik6 variant of IKZF1 gene was much more frequent in adult patients (all P<0.0001). In B-ALL, the existence of Ik6 and that of BCR-ABL were statistically correlated (P<0.0001). In comparison with Western cohorts, the incidence of t(9;22) (q34;q11)/BCR-ABL (14.60%) in B-ALL and HOX11 expression in T-ALL (25.24%) seemed to be much higher in our group, while the incidence of t(12;21) (p13;q22)/ETV6-RUNX1 (15.34%) seemed to be lower in Chinese pediatric patients. The occurrence of hyperdiploidy was much lower either in pediatric (10.61% vs 20-38%) or adult patients (2.36% vs 6.77-12%) in our study than in Western reports. In addition, the frequencies of HOX11L2 in adult patients were much higher in our cohort than in Western countries (20.69% vs 4-11%). In general, it seems that Chinese ALL patients bear more adverse prognostic factors than their Western counterparts do.

Newly diagnosed acute lymphoblastic leukemia in China (II): prognosis related to genetic abnormalities in a series of 1091 cases.

The molecular characterization of cytogenetic abnormalities has not only provided insights into the mechanisms of leukemogenesis but also led to the establishment of new treatment strategies targeting these abnormalities and thereby further improve the prognosis of patients. We analyzed the prognosis of 1091 Chinese patients with newly diagnosed acute lymphoblastic leukemia (ALL) and explored the prognostic impacts of a large number of cytogenetic/molecular abnormalities. It was demonstrated that, in both B- and T-ALL settings, the prognosis was negatively correlated to the age as reported to date. For childhood T-ALL patients, it was also documented that the HOX11 expression represented a favorable prognostic factor as it was in adult ones. We identified CRLF2 overexpression as an intermediate-risk marker and Ik6 variant of IKZF1 gene as a high-risk one when stratifying pediatric B-ALL cases according to cytogenetic/molecular risks. We also found that Ik6 variant and CRLF2 overexpression had an important role in dictating the prognosis of Ph-negative patients, which may be useful markers in guiding the treatment of ALL in the future, with tyrosine kinase inhibitors on the other hand reversing the fate of Ph-positive ALL patients.

AML1-ETO9a is correlated with C-KIT overexpression/mutations and indicates poor disease outcome in t(8;21) acute myeloid leukemia-M2.

AML1-ETO fusion gene is generated from chromosomal translocation t(8;21) mainly in acute myeloid leukemia M2 subtype (AML-M2). Its spliced variant transcript, AML1-ETO9a, rapidly induces leukemia in murine model. To evaluate its clinical significance, AML1-ETO9a expression was assessed in 118 patients with t(8;21) AML-M2, using qualitative and nested quantitative reverse transcriptase (RT)-PCR methods. These cases were accordingly divided into the AML1-ETO9a-H group (n=86, positive for qualitative RT-PCR, with higher level of AML1-ETO9a by quantitative RT-PCR) and the AML1-ETO9a-L group (n=32, negative for qualitative RT-PCR, with lower but still detectable level of AML1-ETO9a by quantitative RT-PCR). C-KIT expression was significantly increased in the AML1-ETO9a-H group, as compared with the AML1-ETO9a-L group. Of the 36 patients harboring C-KIT mutations, 32 patients overexpressed AML1-ETO9a (P=0.0209). Clinically, AML1-ETO9a-H patients exhibited significantly elevated white blood cells count, less bone marrow aberrant myelocytes, increased CD56 but decreased CD19 expression (P=0.0451, P=0.0479, P=0.0149 and P=0.0298, respectively). Moreover, AML1-ETO9a overexpression was related to short event-free and overall survival time (P=0.0072 and P=0.0076, respectively). Taken together, these data suggest that AML1-ETO9a is correlated with C-KIT overexpression/mutations and indicates poor disease outcome in t(8;21) AML-M2.

Clinical and cytogenetic features of 508 Chinese patients with myelodysplastic syndrome and comparison with those in Western countries.

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and leukemia progression. Racial differences may exist on clinical pictures and the molecular events leading to MDS, which are heterogeneous. To better define the clinical and cytogenetic features in Chinese patients, a retrospective multicentric study was performed in 508 MDS cases. Compared with Western countries, Chinese patients showed younger age (median: 49 vs 65-73 years), lower percentages of RARS (2.8 vs 6.6-15.3%), and CMML (5.2 vs 11.7-30.6%). Cytogenetically, among 367 cases with evaluable data, abnormal karyotypes were found in 136 cases, including 56 numerical and 80 structural changes. Incidences of single chromosome 5 and 7 abnormalities were lower than those in Western countries (2.2 vs 17.8-42.5%). However, complex cytogenetic aberrations and chromosome translocations were frequently observed and related to poor prognosis. Both multiple chromosome deletions and translocations were detected in advanced subtypes (RAEB and RAEB-T). Analysis of 200 cases revealed a higher incidence of hepatitis-B-virus infection than that in non-MDS population (21.00 vs 9.75%). This study further confirmed: (1) different genetic/environmental backgrounds between Asian and Western MDS populations; (2) a strong predictive value of cytogenetic abnormalities on disease outcome and involvement of genomic instability in leukemia clone development.

Molecular cytogenetic characterization and clinical relevance of additional, complex and/or variant chromosome abnormalities in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by typical morphological manifestation, t(15;17) translocation and active response to all-trans retinoic acid (ATRA) in the great majority of patients. However, a subset of APL cases may present atypical phenotypic, cytogenetic or molecular features at different stages of the disease. The biological and clinical significance of these features sometimes remains obscure. In this study, 284 APL patients were cytogenetically analyzed and precise diagnosis was performed according to the molecular cytogenetic results. Twenty-six APL patients were identified as having additional, complex and/or variant chromosomal abnormalities at diagnosis or at relapse, 16 of them being further analyzed using fluorescence in situ hybridization (FISH) or chromosome painting (CP). Interestingly, some of these chromosomal aberrations were found to be associated with atypical morphology and/or drug response, indicating a genotype-phenotype correlation. Analysis of the complex karyotype may also allow a better understanding of the levels of cellular origin of the leukemogenesis. Examination of the remission induction and survival data showed that the presence of the additional/complex chromosome abnormalities was related to the prognosis in both primarily diagnosed and relapsed patients in this series.

Studies on the clinical efficacy and pharmacokinetics of low-dose arsenic trioxide in the treatment of relapsed acute promyelocytic leukemia: a comparison with conventional dosage.

Twenty cases of patients with relapsed acute promyelocytic leukemia (APL) were entered into this study for evaluating the clinical efficacy and pharmacokinetics of low-dose arsenic trioxide (As2O3). As2O3 was given at a daily dose of 0.08 mg/kg intravenously for 28 days. Pharmacokinetic study was carried out in eight patients. 16/20 (80%) patients achieved CR. The occurrence of some toxic events including gastrointestinal disturbance, facial edema and cardiac toxicity seemed reduced in the low-dose group than those in the standard-dose group. Differentiation changes were observed in peripheral blood, as well as in bone marrow (BM). Pharmacokinetic study showed that the plasma concentration increased soon after administration of As2O3 with the peak values of 1.535-3.424 micromol/l. After infusion, the plasma concentration was around 0.1-0.5 micromol/l. The arsenic concentration of the plasma of BM aspirates 24 h after administration in five patients was close to the level needed for differentiation-inducing effect. The estimated 2-year OS and RFS were 61.55+/-15.79% and 49.11+/-15.09% respectively, with no difference as compared with those in patients treated with conventional dose (P = 0.2865 and 0.7146, respectively). In conclusion, we demonstrated that low-dose As2O3 had the same effect as the conventional dosage and the mechanism of low-dose arsenic seemed to primarily induce differentiation of APL cells.

Feasibility and clinical significance of real-time quantitative RT-PCR assay of PML-RARalpha fusion transcript in patients with acute promyelocytic leukemia.

INTRODUCTION: To study the relationship between the expression level of the PML-RARalpha fusion transcripts and the clinical status and efficiency of the therapy in acute promyelocytic leukemia (APL) patients, we applied a very sensitive and specific real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) system to quantify the dose of PML-RARalpha fusion transcripts in a series of APL patients at distinct disease stages. MATERIALS AND METHODS: A total of 31 APL patients (19 males and 12 females; aged from 8 to 74 years) from eight hospitals in Shanghai were analysed. Real-time Quantitative RT-PCR was used to measure the normalized dose (DoseN) of PML-RARalpha fusion transcripts. RESULTS: A wide range of PML-RARalpha DoseN above 1 x 10(3) was noted in 25 newly diagnosed patients. PML-RARalpha DoseN was significantly decreased after remission induction with ATRA, ATRA/chemotherapy or As2O3 and further reduced after consolidation. The fact that all patients with long disease free survival had a constantly low PML-RARalpha DoseN below 2 x 10(2) and a higher level predicted impending relapse suggests that this value could serve as a 'threshold' for molecular remission. PML-RARalpha DoseN was also of prognostic value in a group of relapsed patients, since good response to As2O3 reinduction was accompanied by a remarkable reduction of fusion transcript level, whereas patients with high PML-RARalpha Dose(N) after the second CR tended to relapse again rapidly. CONCLUSION: These results confirm that real-time RT-PCR assay for PML-RARalpha transcripts in APL patients is useful in reflecting leukemic burden, assessing response to treatment and indicating the ultimate clinical outcome or curability of disease.

Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia.

Recent studies showed that arsenic trioxide (As2O3) could induce apoptosis and partial differentiation of leukemic promyelocytes. Here, we addressed the possible mechanisms underlying these two different effects. 1.0 microM As2O3-induced apoptosis was associated with condensation of the mitochondrial matrix, disruption of mitochondrial transmembrane potentials (DeltaPsim) and activation of caspase-3 in acute promyelocytic leukemia (APL) cells regardless of their sensitivity to all-trans retinoic acid (ATRA). All these effects were inhibited by dithiothreitol (DTT) and enhanced by buthionine sulfoximine (BSO). Furthermore, BSO could also render HL60 and U937 cells, which had the higher cellular catalase activity, sensitive to As2O3-induced apoptosis. Surprisingly, 1.0 microM As2O3 did not induce the DeltaPsim collapse and apoptosis, while 0.1 microM As2O3 induced partial differentiation of fresh BM cells from a de novo APL patient. In this study, we also showed that 0.2 mM DTT did not block low-dose As2O3-induced NB4 cell differentiation, and 0. 10.5 microM As2O3 did not induce differentiation of ATRA-resistant NB4-derived sublines, which were confirmed by cytomorphology, expression of CD11b, CD33 and CD14 as well as NBT reduction. Another interesting finding was that 0.10.5 microM As2O3 could also induce differentiation-related changes in ATRA-sensitive HL60 cells. However, the differentiation-inducing effect could not be seen in ATRA-resistant HL60 sublines with RARalpha mutation. Moreover, low-dose As2O3 and ATRA yielded similar gene expression profiles in APL cells. These results encouraged us to hypothesize that As2O3 induces APL cell differentiation through direct or indirect activation of retinoic acid receptor-related signaling pathway(s), while DeltaPsim collapse is the common mechanism of As2O3-induced apoptosis.

Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients.

Fifty-eight acute promyelocytic leukemia (APL) patients (11 newly diagnosed and 47 relapsed) were studied for arsenic trioxide (As2O3) treatment. Clinical complete remission (CR) was obtained in 8 of 11 (72.7%) newly diagnosed cases. However, As2O3 treatment resulted in hepatic toxicity in 7 cases including 2 deaths, in contrast to the mild liver dysfunction in one third of the relapsed patients. Forty of forty-seven (85.1%) relapsed patients achieved CR. Two of three nonresponders showed clonal evolution at relapse, with disappearance of t(15;17) and PML-RARalpha fusion gene in 1 and shift to a dominant AML-1-ETO population in another, suggesting a correlation between PML-RARalpha expression and therapeutic response. In a follow-up of 33 relapsed cases over 7 to 48 months, the estimated disease-free survival (DFS) rates for 1 and 2 years were 63.6% and 41.6%, respectively, and the actual median DFS was 17 months. Patients with white blood cell (WBC) count below 10 x 10(9)/L at relapse had better survival than those with WBC count over 10 x 10(9)/L (P =.038). The duration of As2O3-induced CR was related to postremission therapy, because there was only 2 of 11 relapses in patients treated with As2O3 combined with chemotherapy, compared with 12 of 18 relapses with As2O3 alone (P =.01). Reverse transcription polymerase chain reaction (RT-PCR) analysis in both newly diagnosed and relapsed groups showed long-term use of As2O3 could lead to a molecular remission in some patients. We thus recommend that ATRA be used as first choice for remission induction in newly diagnosed APL cases, whereas As2O3 can be either used as a rescue for relapsed cases or included into multidrug consolidation/maintenance clinical trials.

Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF-RARalpha and NPM-RARalpha.

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARalpha and one of four fusion partners: PML, PLZF, NPM, and NuMA genes. To study the leukemogenic potential of the fusion genes in vivo, we generated transgenic mice with PLZF-RARalpha and NPM-RARalpha. PLZF-RARalpha transgenic animals developed chronic myeloid leukemia-like phenotypes at an early stage of life (within 3 months in five of six mice), whereas three NPM-RARalpha transgenic mice showed a spectrum of phenotypes from typical APL to chronic myeloid leukemia relatively late in life (from 12 to 15 months). In contrast to bone marrow cells from PLZF-RARalpha transgenic mice, those from NPM-RARalpha transgenic mice could be induced to differentiate by all-trans-retinoic acid (ATRA). We also studied RARE binding properties and interactions between nuclear corepressor SMRT and various fusion proteins in response to ATRA. Dissociation of SMRT from different receptors was observed at ATRA concentrations of 0.01 microM, 0.1 microM, and 1.0 microM for RARalpha-RXRalpha, NPM-RARalpha, and PML-RARalpha, respectively, but not observed for PLZF-RARalpha even in the presence of 10 microM ATRA. We also determined the expression of the tissue factor gene in transgenic mice, which was detected only in bone marrow cells of mice expressing the fusion genes. These data clearly establish the leukemogenic role of PLZF-RARalpha and NPM-RARalpha and the importance of fusion receptor/corepressor interactions in the pathogenesis as well as in determining different clinical phenotypes of APL.

Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells.

Recent clinical studies in China showed that As2O3 is an effective and relatively safe drug in the treatment of acute promyelocytic leukemia (APL). We found previously that As2O3 can trigger apoptosis of APL cell line NB4 cells, which is associated with downregulation of bcl-2 gene expression and modulation of PML-RAR alpha chimeric protein. To further understand the mechanisms of this alternative therapy for APL, we investigated in this report the effects of a wide range of concentrations of As2O2 on cultured primary APL cells, all-trans retinoic acid (ATRA)-susceptible (NB4 cells) and ATRA-resistant (MR2 subclone) APL cell lines. The results indicated that As2O3 had dose-dependent dual effects on APL cells: inducing preferentially apoptosis at relatively high concentrations (0.5 to 2 micromol/L) and inducing partial differentiation at low concentrations (0.1 to 0.5 micromol/L). The rapid modulation and degradation of PML-RAR alpha proteins, which was induced by As2O3 at 0.1 to 2 micromol/L, could contribute to these two effects. Bone marrow and peripheral blood examination showed that myelocyte-like cells, probably as a result of partial in vivo differentiation, and degenerative cells increased after 2 to 3 weeks of continuous in vivo As2O3 treatment when leukemic promyelocytes decreased. In conclusion, combination of induction of apoptosis and partial differention could be the main cellular mechanisms of As2O3 in the treatment of APL, and PML-RAR alpha could play an important role in determining the specific effects of As2O3 on APL cells.

Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients.

The therapeutic effect of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL) was evaluated among 15 APL patients at relapse after all-trans retinoic acid (ATRA) induced and chemotherapy maintained complete remission (CR). As2O3 was administered intravenously at the dose of 10 mg/d. Clinical CR was achieved in nine of 10 (90%) patients treated with As2O3 alone and in the remaining five patients treated by the combination of As2O3 and low-dose chemotherapeutic drugs or ATRA. During the treatment with As2O3, there was no bone marrow depression and only limited side effects were encountered. Pharmacokinetic studies, which were performed in eight patients, showed that after a peak level of 5.54 micromol/L to 7.30 micromol/L, plasma arsenic was rapidly eliminated, and the continuous administration of As2O3 did not alter its pharmacokinetic behaviors. In addition, increased amounts of arsenic appeared in the urine, with a daily excretion accounting for approximately 1% to 8% of the total daily dose administered. Arsenic contents in hair and nail were increased, and the peak content of arsenic could reach 2.5 to 2.7 microg/g tissue at CR. On the other hand, a decline of the arsenic content in hair and nail was observed after withdrawal of the drug. We conclude that As2O3 treatment is an effective and relatively safe drug in APL patients refractory to ATRA and conventional chemotherapy.