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.

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.

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.

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.

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.

Heterogeneous somatic hypermutation status confounds the cell of origin in hairy cell leukemia.

Hairy cell leukemia (HCL) is thought to arise from a post-germinal center (GC) B-cell, however the exact normal counterpart remains unclear. We performed VH gene analysis of 32 HCL cases, revealing somatically mutated VH genes (<98% homology) in 27 cases and unmutated VH genes in five cases, four of which displayed germline VH genes. Intraclonal heterogeneity was evident in the majority of eight mutated HCLs investigated, although at a lower level compared to GC-derived lymphomas. A novel finding of preferential VH3-30 gene usage was detected (19% of HCLs). Our data confounds the postulated post-GC origin in HCL considering (1) the finding of unmutated HCLs, generally correlating with a pre-GC origin, and (2) the presence of intraclonal variation in mutated HCLs. The latter suggests that the transformed B-cell was frozen when it still had an active mutation process, implying a closer relation to the GC than previously assumed. Furthermore, restricted VH3-30 usage indicates that antigen selection could be a promoting factor in HCL development.

Patients with chronic lymphocytic leukemia with mutated VH genes presenting with Binet stage B or C form a subgroup with a poor outcome.

BACKGROUND AND OBJECTIVES: The immunoglobulin VH gene mutation status is a strong prognostic indicator in B-cell chronic lymphocytic leukemia (CLL), since unmutated VH genes are correlated with short survival. However, the traditional cut-off level dividing mutated and unmutated cases, i.e. more or less than 2% mutations, has been questioned and other cut-offs have been suggested. We investigated whether an alternative cut-off should be applied and the relation of mutational status to another prognostic marker, Binet staging. DESIGN AND METHODS: VH gene mutation status was assessed in 332 CLL cases by polymerase chain reaction amplification and nucleotide sequencing and was further correlated with overall survival using different VH mutation cut-offs (1-7%) and Binet stage. RESULTS: After testing different mutation borders, the 2% cut-off remained the best discriminative level for determining prognosis. Interestingly, prognostic stratification was improved by combining the information on VH gene mutation status with that of Binet stage: unmutated cases (all stages, n=151, mutated cases with stage A (n=77), and mutated cases with stage B or C (n=37) had a median survival of 82, 179 and 74 months, respectively. INTERPRETATION AND CONCLUSIONS: CLL cases displaying mutated VH genes with Binet stage B or C had a survival similar to that of unmutated cases and significantly shorter than that of mutated stage A CLL. Our result reveals clinical heterogeneity within the VH mutated CLL group by inclusion of Binet stage data, a finding which is of importance when considering surrogate marker(s) for VH mutation status.

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.

Ex vivo drug and irradiation sensitivities in hypermutated and unmutated forms of chronic lymphocytic leukemia cells.

Several investigators have now established that chronic lymphocytic leukemia (CLL) is not a uniform disease entity, since approximately half of the cases of CLL have undergone immunoglobulin V region (IgV) hypermutation, whereas the other half display unmutated Ig genes. The median survival time of mutated CLL (M-CLL) cases has been shown to be approximately twice as long as that for unmutated CLL (UM-CLL), but no clear explanation for this difference is currently available. In this work, we have investigated a cohort of previously untreated CLL patients, to see whether the ex vivo sensitivities of leukemic cells of 16 UM-CLL patients differ from those of 8 M-CLL patients, using nine different drugs and two types of irradiation. Our results demonstrated very similar ex vivo sensitivities and tumor cell heterogeneity of sensitivity of UM-CLL and M-CLL cells when tested against chlorambucil, 2-chloro-2'-deoxyadenosine, cyclosporin A, cis-platinum(II)diammine-dichloride, doxorubicin hydrochloride, 2-fluoroadenine-9-beta-D-arabinofuranoside, prednisolone sodium succinate, verapamil, vincristine, gamma-irradiation, and UV-irradiation. This indicates that de novo chemo/radiosensitivity cannot explain the survival difference observed between UM-CLL and M-CLL.

Different gene expression in immunoglobulin-mutated and immunoglobulin-unmutated forms of chronic lymphocytic leukemia.

The mutation status of the immunoglobulin heavy chain variable regions (IgVH) has been found to be a good prognostic indicator for B-cell chronic lymphocytic leukemia (CLL) because unmutated VH genes are associated with rapid disease progression and shorter survival time. To study the differences in gene expression between the Ig-unmutated and Ig-mutated CLL subtypes, we performed gene expression profiling on 31 CLL cases and investigated the VH gene mutation status by sequencing. The array data showed that the greatest variances between the unmutated (20 cases) and the mutated (11 cases) group were in expressions of ZAP70, RAF1, PAX5, TCF1, CD44, SF1, S100A12, NUP214, DAF, GLVR1, MKK6, AF4, CX3CR1, NAFTC1, and HEX. ZAP70 was significantly more expressed in the Ig-unmutated CLL group, whereas the expression of all the other genes was higher in the Ig-mutated cases. These results corroborate a recent finding, according to which the expression of ZAP70 can predict the VH mutation status and suggest that RAF1, PAX5, and other differentially expressed genes may offer good markers for differentiating unmutated cases from mutated cases and thus serve as prognostic markers.

V(H)3-21 gene usage in chronic lymphocytic leukemia--characterization of a new subgroup with distinct molecular features and poor survival.

During recent years it has become evident that lymphoproliferative diseases of B-cell origin display preferential immunoglobulin (Ig) variable heavy chain (V(H)) gene usage. For instance, the V(H)1-69 and V(H)4-34 genes were early found to be overexpressed in B-cell chronic lymphocytic leukemia (CLL) and other B-cell lymphomas. The implications of biased V(H) gene usage have been speculated to be a result of stimulation of unknown antigens, which gives increased proliferation of B-cells with certain V(H) gene configuration and consequently higher probability to undergo transformation. Thus, V(H) gene usage may play a role in development of leukemias and lymphomas. Recently, we could confirm the over usage of the V(H)1-69 and V(H)4-34 genes in CLL, but a novel finding was that the V(H)3-21 gene was preferentially utilized in CLL patients with mutated V(H) genes. These V(H)3-21+ Ig rearrangements showed molecular peculiarities such as shorter lengths of the third complementarity determining region (CDR) and had similar amino acid composition of their CDR3s, implicating recognition of the same antigen in individual tumors. Most of the V(H)3-21+ patients also showed a predominance of lambda chain expression and biased usage of 1 specific V(lambda) gene, V2-14. Furthermore, overall survival appeared to correlate with V(H)3-21 usage and, regardless of V(H) gene mutation status, V(H)3-21+ patients had a poor outcome. All in all, it appears that V(H)3-21 gene usage define a new entity of CLL. The remaining question now to be clarified is if antigen(s) actually are involved in the pathogenesis of V(H)3-21+ CLL.

Similar humoral immunity parameters in chronic lymphocytic leukemia patients independent of VH gene mutation status.

Chronic lymphocytic leukemia (CLL) is a clonal B-cell disorder, which has recently been divided into 2 subtypes based on the somatic hypermutation status of the immunoglobulin heavy chain (IgVH) genes. In patients with unmutated tumor cells the survival time is approximately half of that in mutated cases, but the reason for this difference is poorly understood. Since infections are the major cause of mortality in CLL, we investigated the effect of the mutation status on host immunity and proneness to infections in patients with CLL. As expected, the disease progression seemed to be faster and the disease more advanced (Binet B and C) among unmutated patients than in the mutated ones. Surprisingly, no differences in humoral immunity [immunoglobulin G (IgG), IgM, IgA, IgG subclasses, anti-ABO blood group antibodies and mannan-binding lectin (MBL)] or immune responses (Haemophilus influenzae serotype b conjugate vaccination) were detected between these 2 patient groups. Furthermore, UM-patients were not more prone to infections compared to M-patients, and therapy had no impact on the incidence and pattern of infections in either of the patient groups. The current findings within this patient cohort reveal that the worse outcome in the unmutated subgroup is not caused by more severe defects in immunity and increased susceptibility to infections when compared with the hypermutated group. It is thus conceivable that active immunization procedures such as vaccination can successfully be applied on patients with unmutated IgVH gene and advanced disease stage.

A novel B-cell line (U-2932) established from a patient with diffuse large B-cell lymphoma following Hodgkin lymphoma.

Little is known about mechanisms leading to secondary non-Hodgkin lymphomas (NHL) in patients treated for Hodgkin lymphoma (HL). Our aim was to characterise in detail a cell line derived from a diffuse large B-cell lymphoma (DLBCL) that had developed in a patient with relapsing HL. The cell line U-2932 was established from ascites in a patient suffering from DLBCL previously treated for HL with multiple chemotherapy regimens. Characterisation was based on morphology, immunophenotype, Epstein-Barr virus (EBV)-status, IgH gene rearrangement status, tumourigenicity, p53 sequencing, and immunohistochemical expression of p53, BCL-2 and BCL-6. The karyotype was investigated using G-banding, comparative genomic hybridisation (CGH) and spectral karyotype (SKY) analysis. This cell line shows typical morphological features of a DLBCL and grows as colonies in nude mice. It expresses a B-cell phenotype with a somatically hypermutated V(H)4-39 gene and is negative for EBV. The origin of U-2932 was confirmed by demonstrating an identical V(H)4 rearrangement in ascites from the patient. A point mutation of the tumour-suppressor gene p53 was detected in amino acid position 176 and immunohistochemical over-expression of the p53 protein was also demonstrated. U-2932 carries a complex karyotype including high-level amplifications of the chromosomal bands 18q21 and 3q27 and expresses aberrant BCL-2 and BCL-6 immunohistochemically. We were unable to investigate the clonal relationship between the original HL and U-2932. In conclusion, U-2932 is a unique B cell line established from a patient suffering from HL followed by NHL. Overexpression of BCL-2, BCL-6 and p53 may play a role in the tumourigenesis and drug resistance. This cell line may become a useful tool to better understand the mechanisms responsible for development of secondary NHL in patients treated for HL.

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.

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.

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.