Effects of granulocyte-colony stimulating factor and the expression of its receptor on various malignant cells.

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) is extensively used to improve neutrophil count during anti-cancer chemotherapy. We investigated the effects of G-CSF on several leukemic cell lines and screened for the expression of the G-CSF receptor (G-CSFR) in various malignant cells. METHODS: We examined the effects of the most commonly used commercial forms of G-CSF (glycosylated lenograstim and nonglycosylated filgrastim) on various leukemic cell lines by flow cytometry. Moreover, we screened for the expression of G-CSFR mRNA in 38 solid tumor cell lines by using real-time PCR. RESULTS: G-CSF stimulated proliferation (40-80% increase in proliferation in treated cells as compared to that in control cells) in 3 leukemic cell lines and induced differentiation of AML1/ETO+ leukemic cells. Among the 38 solid tumor cell lines, 5 cell lines (hepatoblastoma, 2 breast carcinoma, squamous cell carcinoma of the larynx, and melanoma cell lines) showed G-CSFR mRNA expression. CONCLUSION: The results of the present study show that therapeutic G-CSF might stimulate the proliferation and differentiation of malignant cells with G-CSFR expression, suggesting that prescreening for G-CSFR expression in primary tumor cells may be necessary before using G-CSF for treatment.

Incidence of submicroscopic deletions vary according to disease entities and chromosomal translocations in hematologic malignancies: investigation by fluorescence in situ hybridization.

Submicroscopic deletions of genes in recurrent chromosomal rearrangements occur frequently in hematologic malignancies, but their incidences have not been reported clearly. We investigated the incidence of submicroscopic deletions and their association with specific genetic rearrangements in various hematologic malignancies. A fluorescence in situ hybridization (FISH) study was conducted in 336 patients with acute lymphoblastic leukemia, 223 patients with acute myeloid leukemia, and 79 patients with chronic myelogenous leukemia. The incidence of submicroscopic deletions in patients with chromosomal rearrangements was the highest in the TEL/AML1 rearrangement (65.0%), followed by BCR/ABL (10.9%), MLL (5.6%), AML/ETO (4.0%), and PML/RARA (0.0%). Submicroscopic deletion was quite common, and incidences were variable according to disease entities and chromosomal translocations. To detect submicroscopic deletions, careful FISH study should be included for the cytogenetic study of hematologic malignancies, and their association with clinical prognosis needs to be further studied.

Application of high throughput cell array technology to FISH: investigation of the role of deletion of p16 gene in leukemias.

Bio-cell chip is a chip that has hundreds of types of cells arrayed and immobilized on a small slide. To elucidate the role of deletion of the p16 gene in hematologic malignancies, the bio-cell chip technique was applied to fluorescent in situ hybridization (FISH) study. We made a bio-cell chip with bone marrow specimen from 109 patients with acute lymphoblastic leukemia (ALL), 102 patients with acute myelogenous leukemia (AML), 47 patients with chronic myelogenous leukemia (CML), and 25 patients with multiple myeloma (MM). A glass slide with 96 separated areas was fabricated, onto which was added methanol/acetic acid fixed cell suspensions for high-throughput FISH for p16. With the successful application of bio-cell chip technique, we found that the deletion of p16 contributed to the oncogenesis in acute leukemia, but not in chronic leukemia. In conclusion, the bio-cell chip, a cell version of ultrahigh-throughput technology, was successfully applied to the FISH study, which can be utilized efficiently in the molecular cytogenetic investigation of hematologic malignancies.

MTHFR 677CC/1298CC genotypes are highly associated with chronic myelogenous leukemia: a case-control study in Korea.

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in folate metabolism and DNA methylation. Studies on MTHFR polymorphism in leukemia have largely focused on the protective role of MTHFR polymorphism in acute lymphoblastic leukemia (ALL). We evaluated the C677T and A1298C polymorphisms using the TaqMan allelic discrimination assay in various malignancies. The study population included 115 subjects with chronic myelogenous leukemia (CML), 200 with acute myelogenous leukemia (AML), 196 with multiple myeloma (MM) and 434 healthy control subjects. The frequency of 1298CC was statistically significantly higher in subjects with CML than that of the controls (OR=5.12, 95% CI: 1.75-14.9, P-value=.003). Of note, the frequencies of 677CC/1298CC genotype were statistically significantly higher in subjects with CML, AML and MM than that of the controls (OR=8.8, 3.5, 3.83, P-value=.002, 0.036, 0.023, respectively). Our results demonstrate that the MTHFR 1298CC homozygote variant is strongly associated with an increased risk of CML, while MTHFR C677T does not significantly affect the risk of CML. Moreover, we demonstrated that MTHFR 677CC and 1298CC genotype might have combined effect on risk of CML, AML and MM and it is inferred that the A1298C may play a different role in carcinogenesis, depending on the types of organs involved, the types of disease entities and the genotype of C677T.

Identification of 13q deletion, trisomy 1q, and IgH rearrangement as the most frequent chromosomal changes found in Korean patients with multiple myeloma.

The most frequent genetic aberrations in multiple myeloma (MM) are 13q deletions and translocations involving the immunoglobulin heavy chain gene (IGH). There have been no reports on the cytogenetic abnormalities found in Korean patients with MM. We investigated the actual prevalence and prognostic value of cytogenetic changes using fluorescence in situ hybridization (FISH). FISH studies with 12 different specific probes for the regions containing the genes or chromosome regions (13q, 1q, IGH, p53, MLL, p16, CEP 7, CEP 11, and CEP 12) were performed in 128 patients. The most frequent change found was 13q deletion (48%), followed by trisomy 1q (45%), IGH translocation (37%), and trisomy 11 (26%). Among the three different probes used to detect 13q deletion, D13S25 (48/58) was the most sensitive probe compared to RB (43/58) and D13S319 (39/58). Among the patients showing one or more changes by FISH, 75% (82/110) had a 13q deletion, a trisomy 1q, or an IGH translocation. Azotemia, anemia, thrombocytopenia, intramedullary plasmacytosis, and stage were significantly associated with the 13q deletion; serum beta(2)-microglobulin, thrombocytopenia, and intramedullary plasmacytosis were also related to trisomy 1q. The pattern of molecular cytogenetic changes in Korean patients with MM is somewhat different from what has been observed in reported Caucasian populations: 37 versus 50-70% with regard to the IGH translocation. The prevalence of the 13q deletion was similar in Korean and Caucasian populations, 48 versus 30-50%. We suggest that the detection of at least these three genetic changes, 13q- trisomy 1q, and an IGH rearrangement, would be helpful for follow-up of Korean patients with MM.

Deletion of any part of the BCR or ABL gene on the derivative chromosome 9 is a poor prognostic marker in chronic myelogenous leukemia.

To evaluate the prognostic significance of submicroscopic deletions of the ABL or BCR gene associated with t(9;22) in chronic myelogenous leukemia (CML), we investigated the incidence of an ABL or BCR deletion on derivative chromosome 9 using fluorescence in situ hybridization (FISH). FISH was performed using the LSI BCR/ABL dual-fusion translocation probe on bone marrow cells of 86 patients with CML. Of 86 patients, ABL deletion was detected in 13 (15.1%) patients and BCR deletion in 8 patients (9.3%). Patients with ABL deletion showed shorter event-free survival time (EFS) than those without ABL deletion (P = 0.020). Patients with BCR deletion showed significantly short overall survival time (OS; P = 0.039). Patients with ABL and/or BCR deletion (14/86 patients, 16.3%) showed significantly short OS and EFS (median OS, 43.0 months; median EFS, 40.0 months), compared to the patients without any BCR or ABL gene deletions (median OS, 94.0 months; median EFS, 90.0 months; P = 0.041 for OS, P = 0.008 for EFS). All the patients with BCR deletion, except for one, had a concomitant ABL deletion, suggesting that BCR deletion occurs in conjunction with ABL deletion. In patients with ABL deletion only, BCR/ABL rearrangement with b2a2 mRNA type tended to be more frequent than in patients without any deletion of the two genes (P = 0.073). Deletion of any of the BCR or ABL genes on derivative chromosome 9 was associated with both short OS and EFS. We conclude that deletion of not only the ABL gene, but also of the BCR gene, is a poor prognostic marker that indicates rapid disease progression in CML.