Weekly administration of docetaxel in combination with estramustine and celecoxib in patients with advanced hormone-refractory prostate cancer: final results from a phase II study

The objective of this study was to evaluate the efficacy and safety profile of weekly docetaxel, estramustine and celecoxib in patients with advanced hormone-refractory prostate cancer. Forty-eight patients received 35 mg m−2 of weekly docetaxel for 3 out of every 4 weeks, 280 mg of estramustine twice daily on days 1–3, 8–10, 15–17 and 400 mg of celecoxib twice daily until progression or toxicity. Cycles were repeated every 28 days for at least six cycles. Patients were evaluated for response and toxicity. Patients received a median of four cycles (range: 1–9). On an intention-to-treat analysis, prostate-specific antigen (PSA) was decreased greater than 50% in 28 out of 48 patients (overall response rate: 58%, 95% confidence interval (CI): 44–72) and median duration of PSA response was 8.0 months (95% CI: 6.9–9.0). After a median follow-up of 11.3 months, the median time to progression was 7.1 months and the median overall survival was 19.2 months. The most frequent severe toxicity was asthenia (15% of patients), diarrhoea and stomatitis (8% of patients, each). Grade 3/4 neutropenia was reported in two patients. There was a toxic death during the study due to a gastric perforation. Celecoxib with weekly docetaxel and estramustine is an effective and safe treatment for patients with hormone-refractory prostate cancer, but it does not seem to add any benefit to docetaxel

ERG rearrangements and PTEN loss in prostate cancer

Prostate cancer (PrCa) is a highly heterogeneous disease and its prognosis, diagnosis and management are still controversial. ERG rearrangements and PTEN loss are frequent and concomitant events in a subset of PrCa. The aim of this thesis is to analyze the potential use of TMPRSS2-ERG and SLC45A3-ERG rearrangements, along with ERG and PTEN expression in the stratification and prognosis of PrCa patients. TMPRSS2-ERG and ERG mRNA overexpression levels are related to a more aggressive phenotype and could be useful PrCa progression markers. Single TMPRSS2-ERG is associated with low grade PrCa and subsequent development of SLC45A3-ERG results in higher ERG expression. The triple hit (TMPRSS2-ERG, SLC45A3-ERG and PTEN loss) is not found in low grade nor low stage tumor, it is associated with Gleason pattern 4 and T3-4 stage and it defines a group of tumors that should be excluded from watchful waiting and are candidates for intense therapy.El càncer de pròstata (CaPr) és una malaltia altament heterogènia i el seu pronòstic, diagnòstic i gestió són polèmiques. Els reordenaments d’ERG i la pèrdua de PTEN són esdeveniments freqüents i concomitants en un subconjunt de CaPr. L'objectiu d'aquesta tesi és analitzar l'ús potencial dels reordenaments TMPRSS2-ERG i SLC45A3-ERG, juntament amb l'expressió d'ERG i PTEN en l'estratificació i pronòstic dels pacients amb CaPr. Nivells de sobreexpressió de TMPRSS2-ERG i ERG estan relacionats amb un fenotip més agressiu i podrien ser útils marcadors de progressió del CaPr. TMPRSS2-ERG s'associa amb CaPr de baix grau i el posterior desenvolupament de SLC45A3-ERG causa una major expressió d’ERG. El “triple hit” (TMPRSS2-ERG, SLC45A3-ERG i pèrdua de PTEN) no es troba en tumors de baix grau i estadi, s'associa amb patró 4 de Gleason i estadis T3-4, i defineix un grup de tumors que són candidats a teràpia intensa i s'han d'excloure d’espera en observaci

B Cell Restricted Expression of Mutated IKZF3 modulates BCR Signaling and Homing Pathways in a Mouse Model of CLL

International audienceMutation in IKZF3 (AIOLOS), a lymphoid transcription factor with a key role in B cell development and function, has been identified as a putative driver of chronic lymphocytic leukemia (CLL) through large-scale WES studies of CLL patients. Prevalent in ~3% CLLs and associated with fludarabine resistance, mutated IKZF3 has been detected uniquely as a hotspot mutation (L162R), localized within the DNA binding domain. The functional effects exerted by this mutation on lymphomagenesis remain unexplored, and were the subject of the current study. We generated a B-cell restricted knock-in mouse line by crossing mice with the Ikzf3 mutant floxed allele with Cd19-Cre animals (i.e. Cre recombinase expression under the Cd19 promoter), and established cohorts carrying either heterozygous mutant-Ikzf3 (Ikzf3Het), homozygous mutant-Ikzf3 (Ikzf3Homo) or wild-type Ikzf3 (Ikzf3WT). We thereafter monitored the animals for leukemia development by serial flow cytometry analyses of peripheral blood. Notably, we detected appearance of ~10-50% clonal B220+CD5+Igk+in 8 of 30 (27%) of the Ikzf3Het mice by 20 months of age, but not in age-matched Ikzf3WTmice (n=30). The cohort of Ikzf3Homo mice has been monitored thus far for 18 months, with identification already of one of 30 (3%) with leukemia with a ~15 months onset. Across animals with CLL-like disease, the pattern of disease localization was consistently observed as detected in the spleen but not in the bone marrow, suggesting preferential homing of Ikzf3-mutant cells to the splenic microenvironment. To dissect the mechanisms underlying Ikzf3-mediated leukemogenesis, we asked whether presence of mutated-Ikzf3 was associated with changes in B cell transcriptional programs in young mice without disease. We performed RNA-sequencing using splenic B cells from Ikzf3Het, Ikzf3Homo or Ikzf3wt animals (3 mice/group, 3-month old). The most striking changes were identified between Ikzf3Homo to Ikzf3wt mice, with ~1400 differentially expressed genes in Ikzf3Homocells compared to Ikzf3wt (>2 fold, adjusted p<0.05). Within this Ikzf3Homo-specific signature, we identified genes regulating B cell positioning within the light zone of germinal centers (GCs, p<0.0001) and BCR signaling pathway members (p<0.05), by gene set enrichment analysis (GSEA). The enhanced activation of the BCR pathway was further confirmed by western blot analysis of splenic B cells stimulated with anti-IgM, with Ikzf3Homo and Ikzf3het mice showing upregulation of phosphorylation in SYK, AKT and ERK after BCR engagement, as compared to Ikzf3WT. Allo-immunization with the T-cell dependent antigen sheep red blood cells (SRBC) for 10 days elicited a higher rate of germinal centers (GC) formation in Ikzf3Homo mice than Ikzf3WT (p<0.01, ANOVA with Tukey's correction), further indicating an enhanced response to BCR stimulation in presence of the mutation. Overall, phenotypic changes of Ikzf3het appeared less pronounced than Ikzf3Homo, in young mice. To further define the functional effects of mutant-Ikzf3 in transcriptional regulation, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using anti-AIOLOS antibody in Ikzf3WT and Ikzf3Homo B cells. While we observed no significant changes in the conserved DNA binding motif or the genomic localization of AIOLOS binding sites, stronger AIOLOS binding at the transcription start sites (TSS) was detected in Ikzf3Homo versus Ikzf3WTcells (p=1.3747x10-5, Student's t-test). These results indicate an enhanced binding of mutated AIOLOS to DNA which may in turn dysregulate gene expression. As an example, upregulation of the chemokine receptor Cxcr4 in Ikzf3Homo B cells was associated with an enrichment of mutant AIOLOS at its promoter region. Functionally, we confirmed that such upregulation was associated with increased migration of Ikzf3Homo cells to SDF-1 in vitro, in a transwell-based chemotaxis assay (p<0.01, ANOVA with Tukey's correction). Overall, this novel model provides evidence of an oncogenic role of IKZF3 mutation in CLL. Ikzf3 mutation induces broad transcriptional and functional changes associated with dysregulation of BCR signaling and homing mechanisms, which may favor disease initiation and progression in vivo. Comparative analyses between Ikzf3-mutant murine CLLs and IKZF3-mutant primary samples are ongoing, to further refine the relevance of Ikzf3 in B cell function and lymphomagenesis. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding

FOXO1 down-regulation is associated with worse outcome in bladder cancer and adds significant prognostic information to p53 overexpression

Nuclear FOXOs mediate cell cycle arrest and promote apoptosis. FOXOs and p53 could have similar effects as tumor suppressor genes. In spite of extensive literature, little is known about the role of FOXO1 and its relationship with p53 status in bladder cancer. Expression of FOXO1 and p53 were analyzed by immunohistochemistry in 162 urothelial carcinomas (UC). Decreased FOXO1 expression, p53 overexpression and the combination FOXO1 down-regulation/p53 overexpression were strongly associated with high grade (P=.030; P=.017; P=.004, respectively), high stage (P=.0001; P<.0001; P<.0001, respectively) or both (P=.0004; P<.0001; P<.0001, respectively). In the overall series of cases, p53 overexpression was associated with tumor progression (hazard ratio [HR]=3.18, 95% confidence interval [CI] 1.19-8.48, P=.02), but this association was even stronger if having any alteration in any of the 2 genes was considered (HR=3.51, 95% CI 1.34-9.21, P=.01). Having both FOXO1 down-regulation and p53 overexpression was associated with disease recurrence (HR=2.75, 95% CI 1.06-7.13, P=.03). In the analysis of the different subgroups, having any alteration in any of the 2 genes was associated with progression in low-grade (P=.005) and pTa (P=.006) tumors. Finally, the combined FOXO1 down-regulation/p53 overexpression was associated with disease recurrence specifically in high-grade (P=.04) and in pT1 stage tumors (P=.007). Adding FOXO1 expression to the immunohistochemical analysis of p53 can provide relevant prognostic information on progression and recurrence of bladder cancer. It may be particularly informative on the risk of progression in the more indolent and on the risk of recurrence in the more aggressive tumors

KZF-L162R Mutation Affects Splenic Mature B Cell Development and Alters Expression of Aiolos Target Genes

Abstract Large-scale DNA sequencing efforts in chronic lymphocytic leukemia (CLL) have identified a broad array of putative cancer drivers arising from somatic mutations in this disease, but functional understanding of the impact of these genetic events on CLL onset and progression remains to be elucidated. One such example is mutation in the IKZF3 gene, encoding the zinc finger protein AIOLOS, mutated in ~2% of CLLs and associated with fludarabine-refractory disease. AIOLOS is a lymphoid-restricted transcription factor and a chromatin remodeler that plays an essential role in B cell development and maturation. In CLL, the IKZF3 mutation, also reported in few cases of diffuse large B cell lymphoma and mantle cell lymphoma,targets a highly conserved hotspot (L162R, homologous to murine L161R) that is localized in the 2nd zinc finger of the DNA-binding domain, required for DNA sequence recognition. Given the localization of this hotspot mutation, we hypothesized that it impacts the function of AIOLOS to drive CLL. To characterize the effects of the IKZF3-L162R mutation, we generated a knock-in mouse line that conditionally expresses the point mutation in a B cell lineage context through crossing with Cd19-cre mice, generating mouse lines carrying Ikzf3-L161R as either a heterozygous mutation (Ikzf3-L161RHet), homozygous mutation (Ikzf3-L161RHomo) or wild-type Ikzf3(Ikzf3WT). Given the established role of Aiolos in lymphoid differentiation, we first asked how the mutation impacts B cell development. By flow cytometry, using established markers to detect marrow pro-B, pre-B, transitional and mature B cell populations, or peritoneal B1a and B1b cell populations, no differences in the proportion of cells were observed between Ikzf3WTor Ikzf3-L161RHet. In the spleen, however, the average proportion of marginal zone B cells (B220+CD23+CD21high) was markedly reduced in heterozygousmice compared to wild type mice (6 mice/group: 4.9% vs. 11.5%, p=0.006), while the average proportion of follicular B cells (B220+CD23+CD21-) was increased (76% vs. 63%; p=0.003). Immunohistochemical staining of spleen sections confirmed that the marginal zone area was significantly reduced in Ikzf3-L161RHetmice (p=0.01). In addition, we noted a higher proliferation rate of B cells from Ikzf3-L161RHetmice when stimulated with LPS and IL-4 for 3 days (p=0.01), suggesting that the mutation confers a survival advantage to B cells. Similar analyses in Ikzf3-L161RHomomice are ongoing. By immunofluorescence and immunoprecipitation, neither Aiolos binding with its partners CHD4, SIN3 or HDAC1, nor its cellular distribution were impacted by the mutation. Of note, the total protein level of Aiolos was increased in Ikzf3-L161RHetmice (9 mice/group; p 20) corresponding to DNA binding sites in the promoters of genes such as Rps19, Ogg1, Dusp2, Phf23 or Brfp1 and confident peaks (FC>10) in the anti-apoptotic gene Mcl1 and in genes involved in BCR signaling (i.e.Syk, Pi3kr1, Nfkbid), suggesting that their expression is under the control of Aiolos. Comparison of the expression by qPCR of these 8 genes in splenic B cells from the 3 mouse lines revealed Dusp2, Mcl1, Syk, Nfkbid and Phf23 to be upregulated in Ikzf3-L161RHomoB cells (p<0.05) but not in Ikzf3-L161RHetB cells. These findings suggest that the mutation directly impacts the expression level of Aiolos target genes. The upregulation of Mcl1 expression is particularly relevant in the context of CLL as dysregulation of anti-apoptotic signaling is characteristic of the disease. In conclusion, these data show that Aiolos mutation affects B cell subpopulation ontogeny, inducing a disproportionate abundance of follicular B cells endowed with high proliferative capacity. The mutation impacts Aiolos transcription capacity leading to upregulation of genes belonging to pathways cardinal to CLL development, including BCR signaling and apoptosis. Ongoing studies focus combining RNA-seq and CHIP-seq in mutant B cells, with the aim of identifying the breadth of differential expressed genes and dysregulated cellular pathways in mutant B cells in an unbiased manner. Disclosures Wu: Neon Therapeutics: Equity Ownership

FOXO1 down-regulation is associated with worse outcome in bladder cancer and adds significant prognostic information to p53 overexpression

Nuclear FOXOs mediate cell cycle arrest and promote apoptosis. FOXOs and p53 could have similar effects as tumor suppressor genes. In spite of extensive literature, little is known about the role of FOXO1 and its relationship with p53 status in bladder cancer. Expression of FOXO1 and p53 were analyzed by immunohistochemistry in 162 urothelial carcinomas (UC). Decreased FOXO1 expression, p53 overexpression and the combination FOXO1 down-regulation/p53 overexpression were strongly associated with high grade (P=.030; P=.017; P=.004, respectively), high stage (P=.0001; P<.0001; P<.0001, respectively) or both (P=.0004; P<.0001; P<.0001, respectively). In the overall series of cases, p53 overexpression was associated with tumor progression (hazard ratio [HR]=3.18, 95% confidence interval [CI] 1.19-8.48, P=.02), but this association was even stronger if having any alteration in any of the 2 genes was considered (HR=3.51, 95% CI 1.34-9.21, P=.01). Having both FOXO1 down-regulation and p53 overexpression was associated with disease recurrence (HR=2.75, 95% CI 1.06-7.13, P=.03). In the analysis of the different subgroups, having any alteration in any of the 2 genes was associated with progression in low-grade (P=.005) and pTa (P=.006) tumors. Finally, the combined FOXO1 down-regulation/p53 overexpression was associated with disease recurrence specifically in high-grade (P=.04) and in pT1 stage tumors (P=.007). Adding FOXO1 expression to the immunohistochemical analysis of p53 can provide relevant prognostic information on progression and recurrence of bladder cancer. It may be particularly informative on the risk of progression in the more indolent and on the risk of recurrence in the more aggressive tumors

Enhancer-Mediated Oncogenic Function of the Menin Tumor Suppressor in Breast Cancer

While the multiple endocrine neoplasia type 1 (MEN1) gene functions as a tumor suppressor in a variety of cancer types, we explored its oncogenic role in breast tumorigenesis. The MEN1 gene product menin is involved in H3K4 trimethylation and co-activates transcription. We integrated ChIP-seq and RNA-seq data to identify menin target genes. Our analysis revealed that menin-dependent target gene promoters display looping to distal enhancers that are bound by menin, FOXA1 and GATA3. In this fashion, MEN1 co-regulates a proliferative breast cancer-specific gene expression program in ER+ cells. In primary mammary cells, MEN1 exerts an anti-proliferative function by regulating a distinct expression signature. Our findings clarify the cell-type-specific functions of MEN1 and inform the development of menin-directed treatments for breast cancer

Enhancer-Mediated Oncogenic Function of the Menin Tumor Suppressor in Breast Cancer

While the multiple endocrine neoplasia type 1 (MEN1) gene functions as a tumor suppressor in a variety of cancer types, we explored its oncogenic role in breast tumorigenesis. The MEN1 gene product menin is involved in H3K4 trimethylation and co-activates transcription. We integrated ChIP-seq and RNA-seq data to identify menin target genes. Our analysis revealed that menin-dependent target gene promoters display looping to distal enhancers that are bound by menin, FOXA1 and GATA3. In this fashion, MEN1 co-regulates a proliferative breast cancer-specific gene expression program in ER+ cells. In primary mammary cells, MEN1 exerts an anti-proliferative function by regulating a distinct expression signature. Our findings clarify the cell-type-specific functions of MEN1 and inform the development of menin-directed treatments for breast cancer

ERG overexpression plus SLC45A3 (prostein) and PTEN expression loss: Strong association of the triple hit phenotype with an aggressive pathway of prostate cancer progression

TMPRSS2 and SLC45A3 rearrangements may coexist in the same tumor. ERG rearrangements and PTEN loss are concomitant events in prostate cancer (PrCa), and can cooperate in progression. We have reported that mRNA expression of TMPRSS2-ERG and SLC45A3-ERG rearrangements plus PTEN loss define an aggressive tumor subset. The aim of this study has been to validate these results by immunohistochemistry in a large cohort of tumors. ERG, SLC45A3 and PTEN immunostaining and their association with pathological features and PSA progression-free survival were analyzed in 220 PrCa (PSMAR-Biobank, Barcelona, Spain). ERG protein expression was found in 46.8% and SLC45A3 and PTEN loss in 30% and 34% tumors, respectively. Single ERG positive immunostaining was associated with GS = 6 tumors (p = 0.016), double ERG+/PTEN loss with GS = 7 (p = 0.008) and Grade Group 2 (GG) or GG3 cases (p = 0.042), ERG+/SLC45A3 loss/PTEN loss ("triple hit") with GS ≥ 8 (p < 0.0001) and GG4 or GG5 tumors (p = 0.0003). None of GS = 6 nor = GG1 cases showed this combination. In the GS ≥ 8 group, ERG+ (p = 0.002), PTEN loss (p = 0.009) and "triple hit" (p = 0.003) were associated with Gleason pattern 3 component, and single SLC45A3 loss (p = 0.036) with GS ≥ 8 without pattern 3. The number of aberrant events and the triple hit were strongly associated with shorter PSA progression-free survival. In GS = 6 PrCa, single ERG+ was also associated with progression. ERG+ identifies a distinct pathway of PrCa. Additional assessment of PTEN and SLC45A3 adds relevant prognostic information. The triple hit phenotype (ERG+/SLC45A3 loss/PTEN loss) is associated with progression and could be used for patient stratification, treatment and follow-up