Taina Lakkala

Effects of radiation fractionation on four squamous cell carcinoma lines with dissimilar inherent radiation sensitivity.

SummaryThe effect of radiation fractionation was investigated using a new 96-well-plate clonogenic assay in four squamous cell carcinoma lines. Earlier experiments had shown that two of the cell lines (UT-SCC-1A and UM-SCC-14A) were inherently relatively sensitive to irradiation, and two (UM-SCC-1 and UM-SCV-1A) relatively resistant. All of the four carcinomas from which the cell lines were established had poor clinical outcome. The radiation doses were given as a single exposure, or split into two or three equal fractions with a 24-h interval. The two inherently sensitive cell lines showed enhanced survival after radiation fractionation as compared with a single dose, whereas the resistant cell lines did not. The result suggests that both the inherent resistance of cancer cells to irradiation and the repair of sublethal radiation damage may lead to treatment failure, and that shortening of the total irradiation time may overcome cancer cell recovery between fractions in some, but not in all carcinomas.

Two novel human B-cell lymphoma lines of lymphatic follicle origin: Cytogenetic, molecular genetic and histopathological characterisation

Abstract:  Two B‐cell lines, designated as HF‐1 and HF‐4, were characterised. The cell lines have complicated karyotype abnormalities including a 14;18 translocation and an 8q24 breakpoint originating from t(2;8)(p11;q24) (HF‐1) or t(1;8)(p21:q24) (HF‐4). The lines have BCL2 rearrangement and they are positive for CD19, CD20, CD22, CD39. HF‐1 is also positive for IgG, and HF‐4 is positive for IgM and IgD. On Northern blot analyses, the 2.6‐kb and 4.2‐kb transcripts corresponding to the major transcripts of CMYC and BCL2, respectively, were seen. In Western blot as well as in FACS (fluorescence‐activated cell sorting) analysis the presence of BCL2 protein in the both HF‐1 and HF‐4 cells was demonstrated. The cell lines are expected to serve as an important tool in the study of the chromosomal mechanism activating cellular oncogenes, the somatic hypermutation mechanism of antigen‐activated B cells and the apoptosis of B cells.

Comparison of bone marrow high mitotic index metaphase fluorescence in situ hybridization to peripheral blood and bone marrow real time quantitative polymerase chain reaction on the International Scale for detecting residual disease in chronic myeloid leukemia

Real time quantitative polymerase chain reaction analyses allow reliable determination of minimal residual disease in chronic myeloid leukemia. Peripheral blood is the first choice as a source of sample for this analysis, and the International Scale for standardization and reporting of minimal residual disease results should be used. Background Recently, an International Scale was proposed for standardizing BCR-ABL transcript measurements and reporting in the assessment of minimal residual disease by real-time quantitative polymerase chain reaction (RQ-PCR). Here we present the setting up of the International Scale conversion factors for a national laboratory by performing both a cross-analysis of a set of standard samples from a reference laboratory and an analysis of bone marrow and peripheral blood samples at diagnosis (from 32 and 27 patients, respectively). Design and Methods A total of 222 bone marrow and 173 peripheral blood mononuclear cell samples from 96 patients with chronic myeloid leukemia were analyzed with RQ-PCR according to Europe Against Cancer protocols. Additionally, 291 bone marrow samples were analyzed with high mitotic index metaphase fluorescence in situ hybridization (metaphase FISH). Results Major molecular response according to the International Scale in BCR-ABL/GUS transcript levels corresponded to a ratio of 0.035% in peripheral blood and 0.034% in bone marrow, yielding the same conversion factor of 2.86 for both types of sample. Based on metaphase FISH, values of 10%/−1.0 log, 1%/−2.0 log and 0.1%/−3.0 log on the International Scale, corresponded to 13%, 2%, and 0.3% of Philadelphia chromosome positive cells in bone marrow, respectively. Conclusions In conclusion, conversion factors can be determined either by cross-analyzing a number of samples with a laboratory that has already established the International Scale or utilizing sufficient numbers of reference samples from chronic myeloid leukemia patients at diagnosis, or using the upcoming international standards.

Sublethal damage repair after fractionated irradiation in endometrial cancer cell lines tested with the 96-well plate clonogenic assay

Two long-established and seven newly established endometrial adenocarcinoma cell lines were tested for their capacity to repair sublethal damage after fractionated irradiation. Cell survival was determined with the 96-well plate clonogenic assay based on limiting dilutions. Total radiation doses of 0.75 Gy, 1.25 Gy, 2.50 Gy, 5.00 Gy and 7.50 Gy were used either as a single dose or divided into two or three equal fractions with a 24 h interval. Survival data were fitted to the linear quadratic model, and the area under the survival curve (AUC), equivalent to the mean inactivation dose, was obtained with numerical integration. The amount of sublethal damage repair (SLDR) was expressed as an area-under-the-curve (AUC) ratio comparing survivals from fractionated-dose with those from single-dose experiments. SLDR capacity of the cell lines expressed as an AUC ratio varied between 1.00 and 1.59, and the mean was 1.17. Two highly radiosensitive cell lines were found to be SLDR-deficient, but most of the cell lines studied had some SLDR capacity. We have earlier shown that endometrial cancer cell lines as a group are more radiosensitive than squamous-cell carcinoma (SCC) lines. Data obtained in this study suggest that the capacity for SLDR in these cell lines is rather limited compared with the majority of SCC lines tested. This finding underlines further the high radioresponsiveness of endometrial cancer.

ETS‐related gene ERG expression in AML patients is significantly associated with NPM1 mutation status

To the Editor: High ERG expression in acute myeloid leukemia (AML) patients has gained considerable attention because it was proposed as an adverse prognostic marker in normal karyotype patients (1–5). This suggests that the high ERG AML subgroup may need an intense treatment protocol. However, little data of ERG expression in AML patients of other cytogenetic groups have been presented (4, 6, 7). We measured bone marrow (BM) ERG expression in 96 AML patients, compared it with NPM1 and FLT3 mutation statuses in cytogenetically defined prognostic groups, and investigated its impact on 3-yr overall survival. Ninety-six consecutive AML patients (55 females, 41 males) in Turku University Central Hospital were included in this study. The numbers of patients in different age groups were 1–16 yr, 7; 17–39 yr, 24; 40–65 yr, 43; 66–79 yr, 22. The treatment of AML patients in different age groups has been described previously (8). In addition, 18 control BM samples were taken from patients suspected to have a haematological malignancy but diagnosed later to suffer of non-malignant haematological diseases, infection-derived cytopenia (n = 10), thrombocytopenia (n = 3), iron-deficiency anaemia (n = 1), mild hypoplasia (n = 4). The patients were cytogenetically classified into three prognostic groups: favourable patients (n = 20) with t(15;17), t(8;21) or inv(16); adverse (n = 17) with )5 ⁄ 5q or )7 or 3q abnormalities or complex karyotype with five or more aberrations, and intermediate (n = 59) comprising other patients. Normal karyotype was found in 44 intermediate risk patients. Mononuclear cells were extracted from BM using Ficoll-Paque gradient centrifugation (8). The ERG ⁄ABL1 expression ratio in these cells was analysed by quantitative PCR as described by Marcucci et al. (1). Methods for FLT3 and NPM1 mutation analyses have been described previously (8). Median and range ERG ⁄ABL1 values for favourable, adverse and intermediate prognostic patients and for controls were 7.1 (2.6–15.6), 7.1 (0.4–17.6), 4.4 (0.05– 15.9) and 4.1 (1.3–12.8), respectively. The AML prognostic groups differed significantly in Kruskal–Wallis test (P = 0.012), but ERG expression did not differ between AML patients and controls (P = 0.23, Mann–Whitney test). FLT3 length mutation was found in 22 ⁄ 96 AML patients and point mutation in seven cases. NPM1 mutation was detected in 27 ⁄ 96 patients. Twenty-six of these were intermediate risk patients, and all except one had normal karyotype. ERG expression differed significantly between NPM1 mutated and unmutated patients in the complete cohort (P < 0.001, Fig. 1) and also among the normal karyotype patients (P = 0.049). The difference of ERG between FLT3 length mutation positive and negative patients was not significant (P = 0.21). When the patients were divided into low and high ERG groups using the median BM ERG ⁄ABL1 ratio (5.12) as the cut-off, AML patients with low ERG expression had significantly higher white blood cell counts (P = 0.028, Mann–Whitney test) and significantly higher frequency of mutations in the NPM1 gene (P < 0.001, Chi-square test). Distribution of mutated NPM1 into low ⁄high ERG subgroups in FAB classes was M1, 8 ⁄ 0 (Binomial test, P = 0.005); M2, 3 ⁄ 4; M4, 5 ⁄ 0 (P = 0.02); M5, 5 ⁄0 (P = 0.02); other, 1 ⁄ 1. Contrary to NPM1 mutation status, FLT3 mutation status did not show any association with ERG expression in any of the morphological subclasses. Three-year overall survival data were available from all patients. Although low ERG was significantly associated with mutated NPM1 (Fig 1), there was no effect of ERG on outcome of patients either in the NPM1 positive (P = 0.87, n = 25) or in the NPM1 negative (P = 0.34, n = 19) normal karyotype patient groups (Kaplan-Meier statistics with log-rank test). In contrast to the previous studies (1–5), no significant prognostic value of ERG expression could be observed among normal karyotype patients (P = 0.33). Supposing that high ERG expression predicts worse outcome, our finding of its preferential existence in the NPM1-unmutated patients is in accordance with the previously reported inferior prognosis of this patient group. However, we could not confirm the previous observation (2) on the association of high ERG expression with worse outcome in normal karyotype AML patients with NPM1 mutation. Being a normal regulator of haematopoiesis (9), ERG may vary during changes in stem cell differentiation. It is also possible that ERG expression may rise in connection to non-neoplastic haematological conditions as in our control group. In conclusion, pretreatment ERG expression is roughly similar and overlapping in AML patients and in patients with non-neoplastic haematological diseases. We observed higher ERG expression in NPM1-unmutated patients than in NPM1-mutated patients. The level of ERG expression had no independent effect on the survival in normal karyotype AML patients. doi:10.1111/j.1600-0609.2010.01483.x European Journal of Haematology 85 (361–362)