P53 Gene Deletion Research Articles

Abstract 4534: A novel and potent small molecule antagonist of HDM2, SCH 1450206, activates the p53 pathway with mechanism-based activity

Abstract p53 is an attractive therapeutic target in oncology since aberrant regulation of p53 expression is associated with cancer development and progression. HDM2 is a primary negative regulator of p53 function in cells. The blockade of the p53-HDM2 interaction presents an attractive approach for development of drugs against tumors expressing wild-type p53. Here we report a novel and potent small molecule antagonist of HDM2, SCH 1450206. It binds selectively to the HDM2 protein with high affinity, with weak affinity to HDMX protein. SCH 1450206 induced dose- and time-dependent stabilization of p53 protein and its downstream targets in several human cancer cell lines with wild-type p53, but not in cell with p53 gene deletion. SCH 1450206-induced stabilization of p53 led to distinct changes in expression of p53 target genes regulating apoptosis and cell cycle checkpoints. Inhibition of cell proliferation and induction of apoptosis were surveyed in a panel of human tumor cell lines following SCH 1450206 treatment. Inactivation of p53 by shRNA in cell lines expressing wt-p53 abolished the inhibition of cell proliferation and appearance of senescence in response to SCH 1450206, suggesting the cellular activity of SCH 1450206 is p53 specific and mechanism-based. Taken together, our results demonstrated the mechanism of action of SCH 1450206, a novel HDM2 antagonist that can activate the p53 pathway, and offers a potential anti-tumor agent targeting tumors expressing wild-type p53. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4534.

Association of absence of p53 gene and chromosome 13q14 variation with prognosis of primary gastrointestinal lymphoma

Objective To assess the role of p53 gene and absence of chromosome 13ql4 in prognosis of primary gastrointestinal lymphoma(PGIL). Methods The expressions of p53 gene and chromosome 13ql4 in paraffin sections from 72 patients with PGIL and 30 patients with lymph node reactive hyperplasia were detected by using improved FISH technique. The association of p53 gene and absence of chromosome 13ql4 with PGIL prognosis was analyzed. Results ① The p53 gene deletion in stage Ⅰ～Ⅱ patients was accounted for 30. 2%, which was lower than that in stage ID—IV patients (63. 2% , χ~2 =6. 397,P=0.01l). ② There was significant difference between MALT lymphoma and non-MALT lymphoma as aspect to p53 gene deletion (28. 6% vs 53. 3%, χ~2 =4. 515, P = 0. 034). ③ The average survival time was (39. 25±22. 73) and (40. 33±23. 18) months in MALT lymphoma and stage Ⅰ～Ⅱ patients with or without single gene variation respectively, and was (9. 11 ±5. 95) and (20. 61 ± 18. 90) months in MALT lymphoma and stage Ⅲ～Ⅳ patients with two genes variation, respectively. ④ 13ql4 deletion was not significantly related to location, pathological type and clinical staging of tumor as well as survival time. However, the prognosis was poor when both 13ql4 and p53 deleted. Conclusions There is a high incidence of p53 gene deletion in non-MALT lymphoma and stage Ⅲ ～Ⅳ patients. Those who have p53 gene and chromosome 13ql4 abnormalities are of high degree of malignant, and the average survival time is shorter than patients with or without single gene deletion. Key words: Primary gastrointestinal lymphoma; p53; Prognosis

Abstract B24: Thoc1 is required for mouse prostate tumorigenesis initiated by loss of Rb and p53

Abstract A large percentage of prostate cancers show either mutational inactivation or deregulation of the Rb tumor suppressor gene. Rb mediates its tumor suppressor function through its association with other cellular proteins. Most of the Rb-binding proteins interact with the A/B pocket domain and the C terminal region. Less than 10 proteins are known to interact with Rb N terminal domain, a domain that appears to be required for normal Rb function in vivo. The Thoc1 gene encodes a protein that binds the Rb N terminal domain. We hypothesize that some of pRb functions in vivo could be mediated in part by interaction with pThoc1. Thoc1 protein has been found to be an essential component of the TREX (transcription/export) complex which is important for mRNP biogenesis and physically couples transcription elongation with RNA processing and export. We have previously reported that E1A/Ras transformed MEFs (mouse embryonic fibroblasts) but not normal MEFs were dependent on Thoc1 for their survival. These observations suggest that tumor cells specifically may be dependent on Thoc1 for survival and Thoc1 may play a role in tumorigenesis. To test our hypothesis, we used a mouse model of prostate cancer where prostatespecific deletion of Rb and p53 genes leads to development of metastatic adenocarcinoma. We find that compound loss of Thoc1, Rb and p53 increased the life-span of mice compared to mice with loss of Rb and p53 alone. Histopathological analyses of prostate tissue showed that initiation of tumorigenesis is delayed in the absence of Thoc1. Tumors that do arise in these mice retain expression of Thoc1. These findings indicate that Thoc1 is required for prostate tumorigenesis. Conditional deletion of Thoc1 alone in mouse prostate does not appear to affect normal prostate development. To test whether Thoc1 is relevant to human prostate cancer, we examined expression of Thoc1 protein in matched normal and prostate tumor tissue cores on tissue microarray. Analysis of nearly 600 patient samples reveals that Thoc1 is significantly overexpressed in tumor tissue compared to normal prostate tissue and pThoc1 levels positively correlated with tumor grade and negatively correlated with biochemical recurrence as indicated by elevated PSA (prostate-specific antigen) levels. Taken together the above findings suggest that Thoc1 is synthetic lethal with the genetic and epigenetic alterations in prostate tumor cells, and hence it may be a potential target for prostate cancer therapy. Citation Information: Cancer Res 2009;69(23 Suppl):B24.

Newly acquired chromosome Abnormalities During Course of CLL: a Retrospective Collection Data From 2 Spanish Centers.

Abstract 4384 BackgroundDifferent recurrent cytogenetic abnormalities have shown clear prognostic value in patients with Chronic Lymphocytic Leukemia (CLL). Acquisition of cytogenetic aberrations during evolution of the disease (i.e. clonal evolution) may have clinical implications. Objective of the studyTo analyze the frequency of clonal evolution and its potential clinical consequences. Methods and MaterialsWe retrospectively collect data from 80 patients diagnosed of CLL, who had been made at least two Fluorescence In Situ Hybridation (FISH) studies, with probes for gene ATM (11q22), gene p53 (17p13), 13q14 region and centromeric probe for chromosome 12. Clonal evolution was found in 16 out of 80 cases (20%), 8 male and 8 women, median age 70 years (41-80). Initial FISH was normal in 9 cases, deletion (del) of 13q14 region was observed in 5, and trisomy of chromosome 12 (+12) in 2 cases. Median of previous lines of treatment were 1 (0-6): Chlorambucil 7, Fludarabine (F) 1, Cyclophosphamide (C) 1, FC combination 3, FC plus Rituximab (FCR) 2, Alemtuzumab 3, CVP 2, CHOP 1, autologous stem cell transplantation (SCT) 1, Bendamustine 4. By flow cytometry, 5/9 analyzed cases were CD38-positive and 3/6 were ZAP70-positive. Out of 13 treated patients, complete response (CR) criteria (by 2008 iwCLL guidelines) were reached in 2, and PR in 8. Median duration of response (DR) were 14 months (0-42), and median time to retreatment (TTR) were 18 months (1-60). ResultsAs clonal evolution, FISH abnormalities detected were: del (p53) in 8 cases, del (ATM) in 7 cases (2 associated with p53 deletion); del(13q) in 3 (homozigous in 2 patients). Treatment were given to 12 patients (75%), indicated in 10 for progressive adenopathies (6/7 cases with del(ATM) y 7/8 with del(p53)), 5 for anemia and 4 for thrombocytopenia (all of them had del(p53)), 3 for short LDT and 3 for B symptoms. Rescue regimens were: FC (4), FR (1), FCR (1), Pentostatin+R (1), Alemtuzumab (1), Cyclophosfamide (2), CHOP+R (1), allogeneic SCT (1). 7/12 responded, including 1 case that reached CR. Median DR was 5 months (0-29), with TTR 9 months (1-41). As following lines they had: Alemtuzumab +/- Methylprednisolone (3), Chlorambucil (1), CVP (1), CHOP (1), Rituximab (1), FR (1), Bendamustine (1). After a median follow-up of 9 months (3-60) from clonal evolution, 5/16 had died, after 3-32 months (median 9). 4 of them died because of disease progression and/or infectious complications. 1 patient had a second malignancy, dying from metastatic prostate cancer. ConclusionsThis retrospective series shows the relevance of clonal evolution in CLL. Selection of clones with deletion of ATM or p53 genes frequently associates with adenopathy progression, while p53 deletion seems to correlate more specifically with cytopenias. Treatment-free and overall survival seem to get compromised. Usefulness of performing FISH at regular intervals might warrant further studies. Disclosures:No relevant conflicts of interest to declare.

Visualization and enrichment of live putative cancer stem cell populations following p53 inactivation or Bax deletion using non-toxic fluorescent dyes

Putative cancer stem cell (CSC) populations efflux dyes such as Hoecsht 33342 giving rise to side-populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoecsht 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non- toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (CloP). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress, and proliferation in several cancer cell lines when stained whereas Hoecsht 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin, and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53-/- cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.

2-phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells

AbstractWe studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 μM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

Mitochondrial Genomic Instability in Colorectal Cancer: No Correlation to Nuclear Microsatellite Instability and Allelic Deletion of hMSH2, hMLH1, and p53 Genes, but Prediction of Better Survival for Dukes’ Stage C Disease

Malfunction of mismatch repair (MMR) system and p53 produces nuclear genomic instability and is involved in colorectal tumorigenesis. In addition to a nuclear genome, eukaryotic cells have cytoplasmic genomes that are compartmentalized in the mitochondria. The aims of this study were to detect the mitochondrial genomic instability (mtGI) in colorectal carcinomas, and to explore its relationship with nuclear genetic alterations and its prognostic meaning. Eighty-three colorectal carcinomas with corresponding normal mucosa were analyzed for mtGI, nuclear microsatellite instability (nMSI), and loss of heterozygosity (LOH) of hMSH2, hMLH1, and p53 genes. Mitochondrial and nuclear alterations were examined for mutual correlation and for associations with clinicopathological features and clinical outcomes. Out of 83 cases, mtGI was identified in 23 carcinomas (27.7%), whereas nMSI was detected in 11 (13.3%). Of the 23 cases with mtGI, only two showed nMSI simultaneously. The frequencies of LOH of hMSH2, hMLH1, and p53 were 16.1%, 11.6%, and 65.3%, respectively. There was no significant association between mtGI and these allelic losses. Notably, Dukes' C patients with mtGI had better disease-free and overall survival than those lacking this feature (p = 0.0516 and 0.0313, respectively). Mitochondrial genomic instability occurs with a high frequency in colorectal carcinomas but is independent of nMSI and allelic deletion of hMSH2, hMLH1, and p53 genes. The results suggest that, instead of nuclear MMR system, there might be different mechanisms involving mitochondrial genomic integrity, and mtGI confers a better prognosis in Dukes' C colorectal cancer.

Alterations in gene expression and sensitivity to genotoxic stress following HdmX or Hdm2 knockdown in human tumor cells harboring wild-type p53

While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. Our laboratory has been interested in tumor cells possessing wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. In this study we utilized RNAi to knockdown HdmX or Hdm2 in MCF7 human breast cancer cells, which harbor wild-type p53 and elevated levels of HdmX and Hdm2 then examined gene expression changes and effects on cell growth.Cell cycle and growth assays confirmed that the loss of either HdmX or Hdm2 led to a significant growth inhibition and G1cell cycle arrest. Although the removal of overexpressed HdmX/2 appears limited to an anti-proliferative effect in MCF7cells, the loss of HdmX and/or Hdm2 enhanced cytotoxicity in these same cells exposed to DNA damage. Through the use of Affymetrix GeneChips and subsequent RT-qPCR validations, we uncovered a subset of anti-proliferative p53 target genes activated upon HdmX/2 knockdown. Interestingly, a second set of genes, normally transactivated by E2F1 as cells transverse the G1-S phase boundary, were found repressed in a p21-dependent manner following HdmX/2 knockdown.Taken together, these results provide novel insights into the reactivation of p53 in cells overexpressing HdmX and Hdm2.

Polyploidy in chronic lymphocytic leukemia with p53 deletion detected by fish: a case report

We report a case of chronic lymphocytic leukemia with a characteristic cytogenetics finding detected by fluorescent in situ hybridization. This case has deletion in p53 gene in 50% of interphase nuclei studied in the peripheral blood and polyploidy in 30% of cells. To our knowledge polyploidy is not commonly reported with chronic lymphocytic leukemia patients.

Disruption of a Functional Relationship Between PU.1 and Mir-155 during the Pathogenesis of Chronic Lymphocytic Leukemia (CLL)

Role of small non-coding microRNAs (miR) in hematopoiesis has been recently established by work demonstrating critical importance of miR-223 for granulocytic development (Johnnidis 2008) and miR-155 in lymphocytic differentiation (Thai 2007) in mice. The importance of miR-155 is further supported by several independent studies demonstrating its increased levels in human diffuse large B-cell lymphoma (Eis 2005, Kluvier 2005, Volinia 2006, Lawrie 2007, Rai 2008) and in chronic lymphocytic leukemia (CLL) (Fulci 2007, Marton 2008). PU.1 is ETS family transcription factor involved in myelo-lymphoid differentiation and is directly negatively regulated by miR-155 (Vigorito 2007). Studying primary CLL (N=27) and normal (N=13) peripheral blood mononuclear cells, we found significantly increased miR-155 levels and also demonstrate that PU.1 levels are significantly decreased. Next, we have tested genes that are targets of both miR-155 and PU.1 including CSF1R and FOS and again demonstrated their significantly decreased expression in CLL. Using gene expression profiling of CLL peripheral lymphocyte samples compared to normal controls, we have determined significant downregulation of the PU.1 mRNA and of numerous miR-155 targets previously reported to be required for myelo-lymphoid differentiation. These findings suggest that the relationship between PU.1 and miR-155 becomes disrupted in CLL. This in turn leads to overexpression of miR-155, reduced levels of PU.1 and of numerous miR-155 targets (N=2481 and N=400 respectively) and to inhibition of PU.1 regulated differentiation and proliferation programs. Next, we correlated negative cytogenetic prognostic features (p53 and ATM gene deletion detected by FISH) of the CLL patients with the miR-155 and PU.1 levels, In a relatively small-size patient cohort studied so far, the low ratios of PU.1/miR- 155 levels are similarly distributed among low, intermediate and high risk CLL patients, suggesting that inverse correlation of PU.1 and miR-155 represents a hallmark of CLL and likely represents a primary event during CLL pathogenesis.

Fluorescent in situ hybridization with a panel of probes detects molecular cytogenetic abnormalities in patients with chronic lymphocytic leukemia

To explore the characteristics and prognostic significance of molecular cytogenetic aberrations in Chinese patients with chronic lymphocytic leukemia (CLL) and the significance thereof in diagnosis of CLL. A panel of probes (LSI D13S319, LSI p53, LSI ATM, CEP 12, LSI MYB, and LSI IGHC/IGHV) and interphase fluorescence in situ hybridization (FISH) were used to prospectively detect the cytogenetic abnormalities in 106 CLL patients, 82 males and 24 females, aged 62 (34 - 86). Molecular cytogenetic aberrations were found in 79 of the 106 CLL patients (74.5%) and 35 patients (33.0%) showed more than two kinds of abnormalities. The most frequent abnormality detected was del (13q14) in 48 cases (45.3%), followed by trisomy of chromosome 12 in 27 patients (25.5%), IgH translocation in 25 patients (23.6%), del (17p13) in 17 patients (16.0%), del (11q22) in 11 patients (10.5%) and del (6q23) in 5 patients (4.7%). The Del (13q14) rate of the group younger than 60 was 56.5%, significantly higher than that of the group aged > or = 60 (36.7%, P = 0.033). There was no significant relationship between molecular cytogenetic aberrations and sex and Binet stages (both P > 0.05). Kaplan-Meier survival analysis showed that the survival time was shorter in the patients with p53 or ATM gene deletion. Patients with sole Del (13q14) had longer survival time than those with other abnormalities. The frequencies of the chromosomal abnormalities in Chinese CLL patients are similar to those in Western countries. Panel FISH has greatly increased the sensitivity of cytogenetic analyses. Del (13q14) is the most frequent abnormality in CLL. Molecular cytogenetic aberrations detected with FISH have important prognostic significance in CLL.

P16 alterations increase the metastatic potential of endometrial carcinoma

ObjectiveThe aim of this study was to investigate the role of p16 in tumorigenesis of endometrial carcinoma (EC). MethodsExpression of p16 protein was analyzed using immunohistochemistry. The methylation status of p16 promoter region was determined by methylation-specific PCR. Deletion analysis of the p16 gene was performed by PCR-analyses. ResultsAberrant protein expression of p16 was observed in 18 of 46 (39.2%) ECs and correlated significantly with p16 alterations, including gene deletions in 26 of 46 (56.5%) ECs and promoter hypermethylation in 8 of 46 (17.4%) ECs (p<0.001). A significant increase in the frequency of p16 alterations from early stage (I–II) to advanced stage (III–IV) ECs was observed (p=0.002). There was no significant correlation between p16 protein expression and the clinico-pathological features of EC. The development of metastases correlated significantly with the frequency of p16 alterations: p16 alterations were detected in 14 of 15 (93.3%) PTs with metastases and in only 18 of 31 (58.1%) PTs without metastases (p=0.018). The genetic comparison of 15 primary ECs and their paired metastases revealed that in most of the cases the deleted region of p16 gene remains the same or becomes larger during the progression from primary tumor to its corresponding metastases. ConclusionOur results suggest that p16 alterations and particularly p16 gene deletions in ECs are associated with increased incidence of metastases.

A novel tumor cell line cloned from mutated human embryonic skeletal muscle cells

A novel sarcoma cell line, denominated MS0812, was spontaneously established from mutated human embryonic skeletal muscle. The cell line grew well and adherent in a plastic dish with strong self-renewal capability, the characteristics of growth was similar to the immortalized cell lines as reported. The cell line contained spindleor fibrous-shaped cells that revealed neoplastic and pleomorphic features. The karyotype of MS0812 cells was abnormal and they exhibited hyperploidy and the chromosome number was ranged from 50 to 158. MS0812 was positive for vimentin, the marker for mesenchymal origins, They were also positive for desmin and actin, indicating their skeletal muscle origin. In serum-free medium culture conditions, MS0812 displayed neuronlike morphology and expressed neuron-specific markers. MS0812 were negative for CD31, CD34, AFP, CK18, CK8 and HLA-DR. And Glycogen can be demonstrated by periodic acid-Schiff stain (PAS) in the cytoplasm of the MS0812. Electron microscope showed the surface of cells has many microvilli and process, lipid droplets and glycogen granules were present in the cytoplasm. MS0812 cells could differentiate into lipid droplets-containing adipocytes in vitro. MS0812 was negative for p16 by immunocytochemistry, and p16 gene deletions were identified by PCR. The MS0812 cells resulted in tumors in Balb/C nude mice in vivo (15/15). Microscopically, the main tissue in tumor was fibrosarcoma, chondrosarcoma islets were also found in the tumor tissues, indicating cartilage formation from MS0812 , chondrosarcoma cells were positive for S100 protein, one tumor contained two components, including fibrosarcoma and chondrosarcoma, this pathological presentation was consistent with dedifferentiated chondrosarcoma, so a novel immortalized cell lines with dedifferentiated chondrosarcoma characteristics was established. We established 7 monoclonal cell lines from MS0812, they showed the same pathological appearance in vivo, and we postulate that fibrosarcoma and chondrosarcoma may be monoclonal origin in human. These findings suggested that MS0812 may be a novel tumor cell line with capability of multiple differentiation. It may provide evidence for the theory that cancer originates from stem cells.

The impact of trisomy 12, retinoblastoma gene and P53 in prognosis of B-cell chronic lymphocytic leukemia

Purpose: Routine cytogenetic analysis frequently fails to identify an abnormal clone in B-cell lymphocytic leukaemia (B-CLL) due to poor response to mitogen stimulation. Fluorescence in situ hybridization (FISH) suggest that chromosomal abnormalities occur more frequently, most commonly trisomy 12, retinoblastoma gene deletion (Rb1 gene) and P53 gene deletion.Patients and methods: 30 patients with B-CLL were enrolled in the trial from two centers in Cairo, Egypt during the period May 2000 to January 2002. Karyotyping and FISH assessment for possible chromosomal abnormalities (trisomy 12, Rb1 gene and P53 gene) were done at initial diagnosis. Results of cytogenetic abnormalities were correlated with clinical picture and survival.Results: The median age was 57·4 years (range 40–75). Karyotyping technique showed that no metaphase could be detected in 30%, metaphase with normal karyotyping was observed in 63% and cytogenetic abnormalities were detected in two cases (one trisomy 12 and one deletion in chromosome 13). FISH examination of interphase and metaphase nuclei revealed cytogenetic abnormalities in 15 cases (50%), trisomy 12 in 9 (30%), Rb1 gene deletion in 5 (17%) and P53 gene deletion in 3. At diagnosis, patients with trisomy 12 were significantly associated with advanced stage and absolute lymphocyte count of ≥30,000/mm3. Univariate analysis showed that absolute lymphocyte count ≥30,000/mm3 (p=0·004) and trisomy 12 (p=0·024) were associated with poor progression free survival.Conclusion: Interphase and metaphase FISH studies improve the cytogenetic diagnosis of chromosomal abnormalities in B-CLL. Lymphocytosis and trisomy 12 may be a good indicator of poor prognosis.

Frequent p16 inactivation by homozygous deletion or methylation is associated with a poor prognosis in Japanese patients with pleural mesothelioma

This study examined the p16 expression status and the P16 gene deletion and methylation status in specimens from Japanese patients with malignant pleural mesothelioma (MPM). Immunohistochemical staining for p16 protein and fluorescence in situ hybridization for the P16 gene were performed using specimens from 30 Japanese patients with primary MPM. The methylation status of the P16 gene was examined in 13 patients whose frozen tumor specimens were available using a methylation-specific PCR assay. Among the 30 patients, the loss of p16 protein expression was observed in 24 patients (80.0%). Twenty-one patients had homozygous deletions, and 9 patients retained the P16 gene. None of the patients with P16 homozygous deletions exhibited p16-positive expression, and 3 patients who retained the P16 gene did not exhibit p16-positive expression. Aberrant P16 methylation was present in two patients with an intact P16 gene but without p16 expression. These results suggest that either a homozygous deletion or methylation is responsible for P16 inactivation. Regarding the prognosis, patients with p16-negative expression had a significantly shorter survival time than those with p16-positive expression (P=0.040). Our study showed that P16 inactivation by homozygous deletions or methylation is a frequent event in Japanese patients with MPMs, relating to poor prognosis. Homozygous deletion is the major cause of P16 inactivation, but methylation also lead to the inactivation of P16 when the P16 alleles are retained.

Analysis of p16 gene deletion or mutation in primary malignant tumor of eyelids

目的 探讨肿瘤抑制基因p16基因的改变与眼睑恶性肿瘤发生的关系及其临床病理学意义.方法采用半定量PCR联合SSCP银染法对59例眼睑恶性肿瘤中p16基因外显子2的缺失与突变进行检测.并用图像分析技术对鳞状细胞癌(SCC)、乳头状瘤、鳞状上皮不典型增生进行DNA倍体及其含量分析. 结果 SCC中p16基因缺失为9/23,基底细胞癌(BCC)为1/17,睑板腺癌为0/10,SCC与后两者之间的差异有统计学意义(P<0.05).DNA倍体及含量分析发现,乳头状瘤、鳞状上皮不典型增生与SCC的DNA峰值倍体分布之间的差异有统计学意义(P<0.05).结论p16基因缺失在眼睑SCC发生中可能起一定作用;DNA倍体分析及其含量测定可用作良性、恶性程度的一个参考指标;p16基因检测有助于肿瘤的临床诊断、治疗及估计预后。

Mechanism of Action of SNS-032, a Novel Cyclin Dependent Kinase Inhibitor, in Chronic Lymphocytic Leukemia: Comparison with Flavopiridol.

Cyclin dependent kinases (Cdks) not only drive cell cycle progression, but also control transcription. For example, Cdk7 and Cdk9 phosphorylate specific sites on the C-terminal domain of RNA polymerase II (pol II) to promote transcription initiation and elongation. Our previous experience with flavopiridol, a non-selective pan-Cdk inhibitor, in primary chronic lymphocytic leukemia (CLL) cells demonstrated inhibition of RNA pol II-driven mRNA synthesis, reduction of the expression of short lived anti-apoptotic proteins, and induction of apoptosis in CLL cells in vitro (Blood 106:2513–19, 2005). Here we studied a novel Cdk inhibitor SNS-032, a 2-aminothiazole derivative with potent and selective inhibitory activity against Cdk2, Cdk7 and Cdk9. We hypothesized that similar to flavopiridol, SNS-032 would induce apoptosis in CLL cells through transcriptional inhibition of anti-apoptotic proteins. To test this hypothesis, we first studied toxicity of SNS-032 in primary CLL samples. At the clinically relevant concentration of 0.3 μM, SNS-032 induced 61 ± 13% apoptosis after 24 hr (mean ± S.D., n=19). This occurred in a time- and concentration-dependent manner that plateaued after 10–12 hr. Additional studies demonstrated this was not due to inactivation of SNS-032. A similar amount of apoptosis was induced in samples with a deletion of p53 gene, indicating a p53-independent cell death. This is important since clinical resistance to fludarabine in CLL is associated with loss of p53 function. Peripheral blood mononuclear cells from healthy donors were less sensitive to SNS-032, suggesting SNS-032 is selective against CLL cells. In direct comparisons SNS-032 was more potent than flavopiridol; 0.1 μM SNS-032 induced a similar amount of apoptosis as did 3 μM flavopiridol. Secondly, we investigated the inhibition of transcription by SNS-032. SNS-032 strongly inhibited the phosphorylation of pol II at both the Ser2 and Ser5 sites of its C-terminal domain, which are the substrates of Cdk9 and Cdk7, respectively. Inhibition of pol II function was associated with a decrease in RNA synthesis, measured by the incorporation of [3H]uridine. After 6 hr incubation, 0.1 μM SNS-032 inhibited 80% of RNA synthesis. Again, SNS-032 was about 30-fold more potent than flavopiridol at inhibiting transcription. As the most sensitive targets of transcription inhibitors are mRNAs and proteins with short intrinsic half-lives, we measured the mRNA levels of short-lived anti-apoptotic proteins by real-time RT-PCR. SNS-032 significantly reduced the mRNA levels of Mcl-1, XIAP and Bcl-2. The protein levels of Mcl-1 and XIAP were also reduced accordingly, whereas Bcl-2 protein remained stable, consistent with a longer half-life of the protein. As CLL cells exemplify a biological context of a tumor that is dependent upon the continued expression of anti-apoptotic proteins for survival, we conclude that inhibition of their expression led to the SNS-032-induced cell death. Thus, while the inhibition of transcription induced by both SNS-032 and flavopiridol is associated with apoptosis in CLL cells, SNS-032 is about 30-fold more potent. This may be due to differences in potency against Cdks and/or differential protein binding. These in vitro results provide rationale for a phase 1 trial of SNS-032 administered to patients with advanced B-lymphoid malignancies that is currently ongoing in multiple centers.

Methylation Profiles of p16, p15 and p14 Genes in Korean Patients with Multiple Myeloma.

Dysruption of cell cycle control genes (p16, p15 and p14) is known to be involved in the tumorigenesis of multiple myeloma (MM). We investigated the inactivation status of p16, p15 and p14 genes using promoter methylation study and fluorescent in situ hybridization (FISH) study in patients with MM and analyzed the association of inactivation of those genes and clinical prognosis.MM Promoter methylation study of p16, p14 and p15 gene was done with newly diagnosed 52 patients with by bisulfite modification and methylation specific PCR. Two sets of primers were used for p16 methylation study and one set of primer was used for p15, p14 methylation study. Deletion of p16, p14 and p15 gene was detected by dual color FISH (Vysis, Downers GroveIL, USA). Overall survival was analyzed by Kaplan Meier Method and Cox's proportional hazard model. Methylation of p16, p15 and p14 promotor was detected in 36/52 (69.2%), 15/52(28.8%) and 7/52 (13.5%) patients with MM, respectively. Methylation of any of p16, p15 or p14 promotor was observed in 44/52 (84.6%) of MM patients Deletion of p16, p15 and p14 was detected in only one of the patients with MM. Of note, in the cases (15/52, 28.8%) showing that in cases with two positive methylation specific PCR of the p16 promotor with two sets of primers, significant lower overall survival rate (p<0.036) were observed compared with only one positive methylation specific PCR. Heavy methylation of p16 protomor was a independent predictive variable for overall survival [Hazard Ratio, 3.74; 95% CI, 1.44–9.68, P = 0.007]. Other adverse prognostic factors by univariate analysis were old age (p=0.014), β2-microglobuline (p=0.026), high serum creatinine levels (≥2.0 mg/dL, p=0.014). More than 80% of MM patients showed the methylation of any of p16, p15 or p14 promotor, which suggest the potential applicability of hypomethlyating agents to MM. The promoter methylation of p16, p14 or p15 was a major contributor to the disruption of cell cycle regulation in MM, whereas both the deletion and/or the promoter methylation of p16, p14, and p15 contributed to the disruption of cell cycle regulation in ALL. In our study, methylation of two positive methylation specific PCR of the p16 promotor was an independent adverse prognostic factor in MM. We infer that quantitative methylation study for p16 promotor is helpful for the evaluation of prognosis. [Display omitted]

P141 Do the “new drugs” antagonize the impact of unfavourable cytogenetic markers in multiple myeloma?

Background In our study, we have concentrated on four aberrations in multiple myeloma (MM) malignant plasma cells: deletions of tumor suppressor genes RB (locus 13q14) and p53 (17q13), translocation involving IGH gene t(4;14), and actually focused amplification of CKS1B gene in 1q21 locus. All of these aberrations are known as negative prognostic factors in patients (pts) treated by conventional therapy or stem cell transplantation. Aims The aim of this study is to show if these selected aberrations act as negative prognostic factors also in patients treated by Velcade or by thalidomide, new drugs used in relapsed patients. Methods We have an increasing group of (recently 42) MM patients. Overall clinical characteristics of the patients at the start of treatment: Average age 63.7 years (SD = 9.4), 57 % (24) of men. 12 % (5/42 pts) were in stage IA, 26 % (11/42 pts) were in stage IIA, 60 % (25/42 pts) were in stage IIIA and 2 % (1 patient) was in stage IIIB. 69 % (29/42 pts) were in first relapse, 24 % (10/42 pts) were in second relapse and the other 7 % (3/42 pts) were in third relapse. 85 % (36/42) pts received at least four cycles of therapy. 64 % (27/42) pts reached overall response (OR) during the follow up (average follow up was 11.0 months, SD = 5.3 months). For identification of malignant plasma cells in bone marrow samples, we use cytoplasmic immunoglobulin (cIg) labeling methodology (Ahmann et al. 1998). This method allows us to identify simultaneously monotypic plasma cells by monoclonal antibody fluorescence (anti-kappa or anti-lambda) and to detect chromosomal abnormalities by FISH (cIg-FISH). Results Cytogenetic findings: Deletion of RB gene was found in 57 % (21/37) of successfully examined patients, deletion of p53 gene was in 53 % (17/32) pts, translocation t(4;14) was in 61 % (22/36) pts and amplification of 1q21 was in 65 % (22/34) pts. Statistical comparison of clinical response in patients with or without each aberration: We have found no significant difference when compared overall survival (OS), time to progression (TTP), progression free survival (PFS), duration of response (DOR) and overall response in subgroups of patients with and without of any selected aberration. Summary/Conclusions In our group of patients, no one of monitored aberrations seems to have any impact, neither positive nor negative, on efficiency of used treatment. It is possible that new drugs, Velcade and thalidomide, antagonize the impact of cytogenetic negative prognostic factors in relapsed patients with MM. However, we will continue in this research to reach larger data set to confirm or disconfirm our results. If this data will be confirmed on larger group of patients, it looks necessary to find other chromosomal abnormalities, relevant for prediction of the prognosis in these patients.

Analysis of p16 Gene Mutation, Deletion and Methylation in Patients with Arseniasis Produced by Indoor Unventilated-Stove Coal Usage in Guizhou, China

The aim of this study was to determine p16 gene mutation, deletion, and promoter 5′ CpG island hypermethylation in peripheral blood mononuclear leukocyte of patients with arseniasis as attributed to exposure to indoor unventilated coal stove. The role of the aberrant change of p16 gene in the induction and development of carcinogenesis in endemic arsenisiasis region in China was also examined. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP), multiplex PCR (mPCR), methylation-specific PCR (MSP), and sequencing techniques were performed to detect (1) mutation of the p16 gene exon 2, (2) homozygous deletion of the p16 gene exon 1 and exon 2, and (3) hypermethylation of the promoter CpG island in peripheral blood mononuclear leukocyte of patients with arseniasis. Results showed no mutation was found in exon 2 of p16 gene. The homozygous deletion frequency of p16 gene was 5 and 15% in control and arseniasis patients, respectively. The homozygous deletion occurred mainly in exon 2, with significant deletion frequencies of 9, 13, and 20% in mild, intermediate, and severe arseniasis groups. The significant homozygous deletion frequency was 9 and 39% in noncarcinoma and carcinoma individuals. The positive rate of p16 gene promoter CpG island hyermethylation was 42 and 2% in the exposed group and the control group, respectively. The positive rate was 26, 42, and 50% in mild, intermediate, and severe arseniasis. The marked different positive rate was 22 and 56% in noncarcinoma and carcinoma individuals, respectively. In conclusion, homozygous deletion and hypermethylation of p16 gene may play an important role in the initiation and development of manifestations seen in endemic arseniasis including carcinogenesis.