Epidermal Keratinocyte Depletion during Five Weeks of Radiotherapy is Associated with DNA Double-Strand Break Foci, Cell Growth Arrest and Apoptosis: Evidence of Increasing Radioresponsiveness and Lack of Repopulation; the Number of Melanocytes Remains Unchanged.

During fractionated radiotherapy, epithelial cell populations are thought to decrease initially, followed by accelerated repopulation to compensate cell loss. However, previous findings in skin with daily 1.1 Gy dose fractions indicate continued and increasing cell depletion. Here we investigated epidermal keratinocyte response with daily 2 Gy fractions as well as accelerated and hypofractionation. Epidermal interfollicular melanocytes were also assessed. Skin-punch biopsies were collected from breast cancer patients before, during and after mastectomy radiotherapy to the thoracic wall with daily 2 Gy fractions for 5 weeks. In addition, 2.4 Gy radiotherapy four times per week and 4 Gy fractions twice per week for 5 weeks, and two times 2 Gy daily for 2.5 weeks, were used. Basal keratinocyte density of the interfollicular epidermis was determined and immunostainings of keratinocytes for DNA double-strand break (DSB) foci, growth arrest, apoptosis and mitosis were quantified. In addition, interfollicular melanocytes were counted. Initially minimal keratinocyte loss was observed followed by pronounced depletion during the second half of treatment and full recovery at 2 weeks post treatment. DSB foci per cell peaked towards the end of treatment. p21-stained cell counts increased during radiotherapy, especially the second half. Apoptotic frequency was low throughout radiotherapy but increased at treatment end. Mitotic cell count was significantly suppressed throughout radiotherapy and did not recover during weekend treatment gaps, but increased more than threefold compared to unexposed skin 2 weeks post-radiotherapy. The number of melanocytes remained constant over the study period. Germinal keratinocyte loss rate increased gradually during daily 2 Gy fractions for 5 weeks, and similarly for hypofractionation. DSB foci number after 2 Gy irradiation revealed an initial radioresistance followed by increasing radiosensitivity. Growth arrest mediated by p21 strongly suggests that cells within or recruited into the cell cycle during treatment are at high risk of loss and do not contribute significantly to repopulation. It is possible that quiescent (G0) cells at treatment completion accounted for the accelerated post-treatment repopulation. Recent knowledge of epidermal tissue regeneration and cell cycle progression during genotoxic and mitogen stress allows for a credible explanation of the current finding. Melanocytes were radioresistant regarding cell depletion.

Metastatic colorectal carcinomas with high SATB2 expression are associated with better prognosis and response to chemotherapy: a population-based Scandinavian study.

Background: Survival and response to therapy in patients with metastatic colorectal cancer (mCRC) are very heterogeneous. There is an unmet need for better markers of prognosis and treatment benefit for mCRC patients. The homeobox 2 gene SATB2 has a highly specific expression in colorectal tissue and is associated with better prognosis in non-metastatic CRC.Material and methods: A population-based cohort of 798 mCRC patients was analysed. From primary tumour material, protein expression was assessed by immunohistochemistry. BRAF and KRAS mutation status was also determined. Associations with clinicopathological data, overall and progression-free survival and response to first-line chemotherapy were analysed.Results: Tumour tissue and clinical data were available from 467 patients. SATB2 was strongly expressed in 58% of cases, significantly more in left-sided, low-grade and wild-type BRAF tumours. Patients with high SATB2 tumours had longer overall survival compared with low SATB2 tumours (median 13 vs 8 months respectively, p < .001). Chemotherapy was given to 282 patients (63%). Patients with high SATB2 tumours had longer OS (median 22 vs 15 months respectively, p = .001) and more often responded to chemotherapy than those with low SATB2 (objective response 43% vs 29%, p = .02; clinical response 83% vs 67%, p = .004). Progression-free survival on first-line irinotecan chemotherapy was longer in high SATB2 cases (median 8 vs 4 months respectively, p = .019). Patients with both low SATB2 expression and mutated BRAF (n = 69) had particularly poor survival compared to the rest (median 8 and 12 months respectively, p = .001). In multivariable analysis, the SATB2 findings were independent of known clinicopathological prognostic markers, including BRAF mutation status.Conclusion: Patients with mCRC expressing high level of SATB2 have better prognosis and response to chemotherapy than those with low SATB2 expression. Patients with both low SATB2 expression and mutated BRAF had particularly poor prognosis and could thus benefit from more aggressive therapies.

High BRAF Mutation Frequency and Marked Survival Differences in Subgroups According to KRAS/BRAF Mutation Status and Tumor Tissue Availability in a Prospective Population-Based Metastatic Colorectal Cancer Cohort.

RAS and BRAF mutations impact treatment and prognosis of metastatic colorectal cancer patients (mCRC), but the knowledge is based on trial patients usually not representative for the general cancer population. Patient characteristics, treatment and efficacy according to KRAS, BRAF and MSI status were analyzed in a prospectively collected unselected population-based cohort of 798 non-resectable mCRC patients. The cohort contained many patients with poor performance status (39% PS 2-4) and elderly (37% age>75), groups usually not included in clinical trials. Patients without available tissue micro array (TMA) (42%) had worse prognostic factors and inferior survival (all patients; 7m vs 11m, chemotherapy-treated;12m vs 17m). The 92 patients (21%) with BRAF mutation had a poor prognosis regardless of microsatellite instability, but receipt of 1-2nd chemotherapy was similar to wildtype BRAF patients. Median survival in this cohort varied from 1 month in BRAF mutated patients not given chemotherapy to 26 months in wildtype KRAS/BRAF patients <75 years in good PS. TMA availability, BRAF mutation and KRAS mutation were all independent prognostic factors for survival. The observed 21% BRAF mutation incidence is higher than the previously and repeatedly reported incidence of 5-12% in mCRC. Screening for BRAF mutations before selection of treatment is relevant for many patients, especially outside clinical trials. A BRAF mutation only partly explained the very poor prognosis of many mCRC patients. Survival in unselected metastatic colorectal cancer patients is extremely variable and subgroups have an extremely short survival compared to trial patients. Patients without available TMA had worse prognostic factors and shorter survival, which questions the total generalizability of present TMA studies and implies that we lack information on the biologically worst mCRC cases. Lack of available tissue is an important underexposed issue which introduces sample bias, and this should be recognized more clearly when conclusions are made from translational mCRC studies.

The MDM2 polymorphism SNP309 is associated with clinical characteristics and outcome in diffuse large B-cell lymphoma.

INTRODUCTION: The murine double minute 2 (MDM2) gene encodes a regulatory protein of the p53 pathway. A single nucleotide polymorphism (T to G change) at position 309 (SNP309) in the promotor region of MDM2 affects the transcription activity of MDM2 and has been found to be a negative prognostic marker in several cancers. PATIENTS AND METHODS: In this study, the MDM2 SNP309 polymorphism was analysed in 201 patients with diffuse large B-cell lymphoma and analysed in relation to clinical characteristics and prognosis. RESULTS: Patients homozygous for SNP309T had a significantly longer overall survival, lymphoma-specific survival and disease-free survival (P = 0.002; 0.004 and 0.006 respectively) compared to patients carrying a G allele. The longer overall survival was seen in the subgroup of patients not treated with Rituximab, however, not for Rituximab-treated patients (P = 0.01 and 0.2 respectively). The group homozygous for the T allele also had lower age at diagnosis, a tendency towards lower aaIPI and a significantly lower proportion of patients with p53 aberrations compared to the group including at least one G allele. However, the survival differences persisted even after removal of cases with known p53 aberrations from the analysis. CONCLUSION: Polymorphism in MDM2 SNP309 could be correlated to some clinical characteristics and for patients not treated with immunotherapy, a G allele was correlated to poor survival, whereas no survival differences were found for patients treated with Rituximab. Herewith, we provide additional information about Diffuse large B-cell Lymphoma (DLBCL) biology and highlight the importance of evaluation of molecular markers in relation to treatment.

Low expression of microRNA-129-5p predicts poor clinical outcome in diffuse large B cell lymphoma (DLBCL).

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous group of B cell lymphomas. MicroRNA expression provides a new and interesting tool for understanding the biology and clinical course of DLBCL. The present study presents microRNA-129-5p expression data from DLBCL patients treated with CHOP or R-CHOP. Patients with low microRNA-129-5p expression had a median survival of 23 months and a significantly shorter overall survival (P = 0.0042) compared to patients with high microRNA-129-5p expression, who had a median survival of 58 months. We also found that patients treated with R-CHOP only and displaying low microRNA-129-5p expression had a significantly shorter overall survival compared to patients with high microRNA-129-5p expression; all such patients were still alive at the time of last follow-up (P = 0.043). No significant difference was found among microRNA-129-5p expression in tumor tissue, the tissue surrounding the tumor, and normal controls. To our knowledge, this is the first report to show that the expression of microRNA-129-5p can affect the clinical outcome of DLBCL patients and that microRNA-129-5p may be involved in the biology of DLBCL development, although larger studies are necessary to confirm this. Further investigations may also help to elucidate the biological role of microRNA-129-5p in DLBCL.

The combined effects of single-nucleotide polymorphisms, tobacco products, and ethanol on normal resting blood mononuclear cells.

INTRODUCTION: Tobacco and ethanol consumption are crucial factors in the development of various diseases including cancer. In this investigation, we evaluated the combined effects of a number of single nucleotide polymorphisms (SNPs), with ethanol and tobacco products on healthy individuals. METHODS: Pure nicotine, cigarette smoke extract, and Swedish snuff (snus) extract were used. The effects were examined by means of in vitro cell cycle progression and cell death of peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. RESULTS: After 3 days, in vitro, resting PBMCs entered the S and G2 stage in the presence of 100 µM nicotine. The PBMCs only proceeded to S stage, in the presence of 0.2% ethanol. The nicotine- and ethanol-induced normal cell cycle progression correlated to a number of SNPs in the IL12RB2, Rad 52, XRCC2, P53, CCND3, and ABCA1 genes. Certain SNPs in Caspases 8, IL12RB2, Rad 52, MMP2, and MDM2 genes appeared to significantly influence the effects of EtOH-, snus-, and snus + EtOH-induced cell death. Importantly, the highest degree of cell death was observed in the presence of smoke + EtOH. The amount of cell death under this treatment condition also correlated to specific SNPs, located in the MDM2, ABCA1, or GASC1 genes. CONCLUSIONS: Cigarette smoke in combination with ethanol strongly induced massive cell death. Long-term exposure to smoke and ethanol could provoke chronic inflammation, and this could be the initiation of disease including the development of cancer at various sites.

High expression of microRNA-200c predicts poor clinical outcome in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of B-cell lymphomas. A new and important tool for understanding the biology and clinical course of DLBCL is microRNA expression. This study presents microRNA-200c expression data from 61 DLBCL patients treated with CHOP or R-CHOP. Patients with high microRNA-200c expression had a median survival of 20.3 months and a significantly shorter overall survival (P=0.019) compared to patients with low microRNA-200c expression, who had a median survival of 35.8 months. We also found that patients treated with R-CHOP only and displaying high microRNA-200c expression had a significantly shorter overall survival compared to patients with low microRNA-200c expression, where all patients were still alive at the time of the last follow-up (P=0.0036). Lastly, we found that patients with high microRNA-200c expression had a significantly shorter time from initial diagnosis to the first relapse compared to patients with low microRNA-200c expression (P=0.0001). To our knowledge, this is the first study showing that the expression of microRNA-200c affects the clinical outcome of DLBCL patients, and that microRNA-200c is involved in the biology of DLBCL development, although larger studies are necessary to confirm this. Further investigations may also help to elucidate the biological role of microRNA-200c in DLBCL.

miR-34a expression, cell cycle arrest and cell death of malignant mesothelioma cells upon treatment with radiation, docetaxel or combination treatment.

OBJECTIVE: Malignant mesothelioma (MM) is a highly aggressive tumour related to asbestos exposure. Histopathologically, the tumour is classified as epithelial, sarcomatoid or biphasic. To date, MM is still an incurable disease. METHODS: To evaluate treatment strategies on MM cells, the effects of radiotherapy, docetaxel or a combination of both on MM cells derived from the sarcomatoid type ZL34 and the epithelial type M28K were investigated. The TP53 gene, micro-RNA expression, cell cycle distribution and cell death were assessed as indicators of treatment effects. RESULTS: Despite the normal TP53 gene sequences in these cell lines, radiation-induced miR-34a expression was detected only in the M28K cells. Increasing G0/G1 cell numbers were detected in irradiated M28K and ZL34 cells. There was more radiation-induced cell death in M28K compared to ZL34 cells. The highest degree of cell cycle arrest at G2 and cell death in both cell types was obtained in the presence of docetaxel. The combination of docetaxel and radiation did not show any additive effects on miR-34a expression, cell cycle arrest or cell death in either the M28K or ZL34 cells. CONCLUSION: Microtubule formation and other related functions by docetaxel might be the most suitable treatment modulation in both sarcomatoid and epithelial types of MM.

Mast cell infiltration is a favourable prognostic factor in diffuse large B-cell lymphoma.

Previous studies indicate that the inflammatory response in diffuse large B-cell lymphomas (DLBCL) is important for the clinical outcome. Mast cells are key regulators in this response; we investigated whether the number of tryptase-positive mast cells is correlated with clinical outcome. Patients with many mast cells had a significantly better event-free survival (EFS) compared to those with few mast cells (P < 0.03 in both germinal centre (GC) and non-GC DLBCL. This supports the idea that the infiltration of mast cells is a reflection of the host inflammatory response and is related to a favourable outcome.

Telomere length and correlation with histopathogenesis in B-cell leukemias/lymphomas.

Telomere length was recently reported to correlate with cellular origin of B-cell malignancies in relation to the germinal center (GC). In this report, we measured telomere length by quantitative-PCR in 223 B-cell lymphomas/leukemias and correlated results with immunoglobulin (Ig) mutation status and immunostainings for GC/non-GC subtypes of diffuse large B-cell lymphoma (DLBCL). Shortest telomeres were found in Ig-unmutated chronic lymphocytic leukemia (CLL) [median telomere to single copy gene value (T/S) 0.33], differing significantly to Ig-mutated CLL (0.63). Contrary to this, mantle cell lymphomas (MCLs) exhibited similar telomere lengths regardless of Ig mutation status (0.47). Telomere length differed significantly between GC-like (0.73) and non-GC-like DLBCLs (0.43), and follicular lymphomas (FLs) had shorter telomeres (0.53) than GC-DLBCL. Hairy cell leukemias, which display Ig gene intraclonal heterogeneity, had longer telomeres (0.62) than FLs and non-GC-DLBCL, but shorter than GC-DLBCL. We conclude that although DLBCL and CLL subsets can be clearly distinguished, telomere length reflects many parameters and may not simply correlate with GC-related origin.

Genomic imbalances during transformation from follicular lymphoma to diffuse large B-cell lymphoma.

Follicular lymphoma is commonly transformed to a more aggressive diffuse large B-cell lymphoma (DLBCL). In order to provide molecular characterization of this histological and clinical transformation, comparative genomic hybridization was applied to 23 follicular lymphoma and 35 transformed DLBCL tumors from a total of 30 patients. The results were also compared with our published findings in de novo DLBCL. Copy number changes were detected in 70% of follicular lymphoma and in 97% of transformed DLBCL. In follicular lymphoma, the most common alterations were +18q21 (33%), +Xq25-26 (28%), +1q31-32 (23%), and -17p (23%), whereas transformed DLBCL most frequently exhibited +Xq25-26 (36%), +12q15 (29%), +7pter-q22 (25%), +8q21 (21%), and -6q16-21(25%). Transformed DLBCL showed significantly more alterations as compared to follicular lymphoma (P=0.0001), and the alterations -6q16-21 and +7pter-q22 were only found in transformed DLBCL but not in follicular lymphoma (P=0.02). Alterations involving +13q22 were significantly less frequent, whereas -4q13-21 was more common in transformed as compared to de novo DLBCL (P=0.01 and P=0.02, respectively). Clinical progression from follicular lymphoma to transformed DLBCL is on the genetic level associated with acquisition of increasing number of genomic copy number changes, with non-random involvement of specific target regions. The findings support diverse genetic background between transformed and de novo DLBCL.

Strikingly homologous immunoglobulin gene rearrangements and poor outcome in VH3-21-using chronic lymphocytic leukemia patients independent of geographic origin and mutational status.

We recently reported that Swedish VH3-21-using chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite VH mutation status. To investigate this further, we analyzed the VH and VL gene rearrangements in 90 VH3-21+ patients from Sweden, Germany, Italy, United States, Finland, and Australia and correlated these data with survival and other prognostic markers. Sixty-three percent exhibited mutated VH genes and 37% unmutated VH genes. Fifty (56%) patients displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif DANGMDV. Also, a highly biased Vlambda2-14 use was evident in 72% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although VH3-21+ CLLs have a remarkably predominant lambda expression, analyses of kappa deleting element indicated a conserved light-chain rearrangement order. The overall survival was poor in the VH3-21+ cohort (median survival, 88 months), with no significant difference in relation to mutation status or CDR3 homology. High ZAP-70 and CD38 expression was found in both mutated and unmutated VH3-21+ cases as well as a slight increase of 11q-aberrations. In summary, highly restricted B-cell receptors and worse outcome characterize VH3-21+ CLLs independent of geographic origin and mutation status.