Gaëlle Fromont

Prognostic value of O6-methylguanine-DNA methyltransferase status in glioblastoma patients, assessed by five different methods

This multicenter study assesses the value of O6-methylguanine-DNA methyltransferase (MGMT) status for predicting overall survival in glioblastoma patients. Five methods are used, to identify the approach with the best prognostic value. Eighty-one tumors were obtained from patients with glioblastomas treated by surgery and radiotherapy with concomitant temozolomide (TMZ) followed by adjuvant TMZ. MGMT promoter methylation was assessed by qualitative methyl-specific polymerase chain reaction (MSP), semiquantitative methyl-specific polymerase chain reaction (SQ-MSP), and pyrosequencing, while MGMT expression was measured at the RNA level by quantitative real-time PCR (Q-RT-PCR) and at the protein level by immunohistochemistry (IHC). MGMT promoter methylation as evaluated by MSP, SQ-MSP, and pyrosequencing was significantly correlated with overall survival. The best predictive value was obtained by pyrosequencing of one specific CpG position. Overall survival was 14 and 25xa0months for patients with percentages of methylation below and above the median, respectively. In contrast, MGMT status determined by Q-RT-PCR and IHC showed little or no correlation with overall survival, respectively. These results confirm the prognostic value of MGMT promoter methylation in glioblastoma patients initially treated with TMZ. SQ-MSP allowed better discrimination than classical MSP, and pyrosequencing represented a good option.

Pivotal role of the lipid raft SK3-Orai1 complex in human cancer cell migration and bone metastases

The SK3 channel, a potassium channel, was recently shown to control cancer cell migration, a critical step in metastasis outgrowth. Here, we report that expression of the SK3 channel was markedly associated with bone metastasis. The SK3 channel was shown to control constitutive Ca(2+) entry and cancer cell migration through an interaction with the Ca(2+) channel Orai1. We found that the SK3 channel triggers an association with the Orai1 channel within lipid rafts. This localization of an SK3-Orai1 complex seemed essential to control cancer cell migration. This suggests that the formation of this complex in lipid rafts is a gain-of-function, because we showed that none of the individual proteins were able to promote the complete phenotype. We identified the alkyl-lipid Ohmline as a disrupting agent for SK3-Orai1 lipid raft localization. Upon Ohmline treatment, the SK3-Orai1 complex moved away from lipid rafts, and SK3-dependent Ca(2+) entry, migration, and bone metastases were subsequently impaired. The colocalization of SK3 and Orai1 in primary human tumors and bone metastases further emphasized the clinical relevance of our observations. Targeting SK3-Orai1 in lipid rafts may inaugurate innovative approaches to inhibit bone metastases.

DNA-PKcs expression predicts response to radiotherapy in prostate cancer.

PURPOSEnDouble-strand breaks, the most lethal DNA lesions induced by ionizing radiation, are mainly repaired by the nonhomologous end-joining system. The expression of the nonhomologous end-joining pathway has never been studied in prostate cancer, and its prognostic value for patients undergoing radiotherapy remains unknown.nnnMETHODSnPretreatment biopsies from 238 patients treated with exclusive external beam radiotherapy for localized prostate cancer with ≥ 2 years of follow-up were reviewed to reassess the Gleason score. Of these 238 cases, 179 were suitable for in situ analysis and were included in the tissue microarrays. Expression of the nonhomologous end-joining proteins Ku70, Ku80, DNA-dependent protein kinase, catalytic subunits (DNA-PKcs), and X-ray repair cross complementing 4-like factor was studied by immunohistochemistry, together with the proliferation marker Ki67.nnnRESULTSnThe predictive value of the Gleason score for biochemical relapse (using the Phoenix criteria) was markedly improved after review (P<.0001) compared with the initial score (P=.003). The clinical stage, pretreatment prostate-specific antigen level, and perineural invasion status were also associated with progression-free survival (P=.005, P<.0001, and P=.03, respectively). High proliferation (>4%) tends to be associated with biochemical recurrence; however, the difference did not reach statistical significance (P=.06). Although the expression of Ku70, Ku80, and X-ray repair cross complementing 4-like factor was not predictive of relapse, positive DNA-PKcs nuclear staining was closely associated with biochemical recurrence (P=.0002). On multivariate analysis, only the Gleason score, prostate-specific antigen level, and DNA-PKcs status remained predictive of recurrence (P=.003, P=.002, and P=.01, respectively).nnnCONCLUSIONSnThe results of the present study highly suggest that DNA-PKcs could be a predictive marker of recurrence after radiotherapy, independently of the classic prognostic markers, including the Gleason score modified after review.

8q24 amplification is associated with Myc expression and prostate cancer progression and is an independent predictor of recurrence after radical prostatectomy

Genomic alterations affecting the 8q24 region are frequent in prostate cancer. Together with the oncogene MYC, other genes located in the surrounding of the amplified region could also be candidate targets. Tissue microarrays were constructed with prostate cancer tissues from (1) a case-control population of patients treated by radical prostatectomy (n = 242; 121 cases with biochemical relapse matched with 121 cancers with identical clinicopathologic features but without relapse), (2) castration-resistant disease (n = 55), and (3) metastatic cancers (n = 28). Fluorescence in situ hybridization and immunohistochemistry were used on tissue microarrays and slides to analyze, respectively, the amplification status of 8q24 and protein expression of genes located at 8q24. Amplification at the MYC locus was observed in 29% of cases and was closely associated with both disease progression (from 15% in pT2 tumors to 53% in metastasis; P = .001), and Gleason score (from <3% in Gleason 6 tumors to 66% in Gleason 8 and more tumors; P < .0001). The expression of genes located at 8q24 did not correlated with the amplification status, except for the Myc protein (P = .002). MYC amplification status but not Myc protein expression was significantly predictive of biochemical recurrence after prostatectomy, together with the proliferation marker Ki-67 and independently from known prognostic factors, including TNM stage and Gleason score. The MYC amplification status could constitute a useful prognostic tool for patients treated by radical prostatectomy, particularly for those with dAmico intermediate risk, whose clinical behavior is currently difficult to predict.

Expression of estrogen related proteins in hormone refractory prostate cancer: association with tumor progression.

PURPOSEnDespite increasing evidence that estrogen signaling has a key role in prostate cancer development and progression, few studies have focused on the estrogen pathway in the transition from hormone sensitive to hormone refractory tumors. We investigated the expression of proteins related to androgen and estrogen metabolism in paired prostate cancer samples collected before androgen deprivation therapy and after hormonal relapse.nnnMATERIALS AND METHODSnThe study included 55 patients treated for prostate cancer only with androgen deprivation therapy and in whom tissue was available before treatment induction and after recurrence. Immunohistochemistry was performed using tissue microarray with antibodies directed against androgen receptor, phosphorylated androgen receptor, estrogen receptor α, estrogen receptor β, 5α-reductase 1 and 2, aromatase, BCAR1 and the proliferation marker Ki67.nnnRESULTSnCompared to hormone sensitive samples, tissues collected after hormonal relapse were characterized by increased expression of Ki67, androgen receptor, phosphorylated androgen receptor (p <0.001) and BCAR (p = 0.03), and by lower staining for 5α-reductase 2 (p = 0.002), estrogen receptor β (p = 0.016) and aromatase (p <0.001). Shorter time to hormonal relapse was associated with high expression of aromatase and BCAR1 on diagnostic biopsy, together with low staining for estrogen receptor α in stromal cells. Overall survival was significantly shorter when tissues collected after relapse showed a high proliferation index and low estrogen receptor α expression.nnnCONCLUSIONSnResults revealed dysregulation of proteins involved in androgen pathways, and in estrogen synthesis and signaling during the development of hormone refractory prostate cancer.

Biological significance of perineural invasion (PNI) in prostate cancer.

In order to better understand the biological significance of perineural invasion (PNI) in prostate cancer, we aimed to analyze in situ the expression of molecules involved in tumor growth or nerve trophicity.

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.

Ductal carcinoma of the prostate shows a different immunophenotype from high grade acinar cancer

Ductal carcinoma (DC) of the prostate is an entity distinct from the common acinar cancer (AC), both on clinical and morphological aspects. We aimed to analyze the expression of molecules involved in either hormonal signalling or androgen independent pathways, in DC compared to high grade AC.

BCAR1 expression improves prediction of biochemical reccurence after radical prostatectomy

Because prostate cancer exhibits a great variability in clinical outcome, biomarkers that can be used in daily practice are needed to better stratify patients into prognostic groups. Since steroid hormones play a central role in the development and progression of prostate cancer, we aimed to analyze in a matched nested case–control study the value of molecules involved in steroid signaling, to predict recurrence after radical prostatectomy, independently from standard prognostic tools.

Epithelial Phenotypes in the Developing Human Prostate

An intermediate population has been identified among prostate glands called transiently amplifying (TA) cells, which are characterized by coexpression of basal and luminal cytokeratins (CKs), high proliferation, and lack of p27 expression. These cells are rare in the normal adult prostate and increase in pretumoral conditions, but their importance in the developing gland remains unknown. We analyzed fetal prostates for the expression of CKs (5/6, 18, 19) and factors involved in proliferation and apoptosis: p63, Ki67, p27, epidermal growth factor (EGFR), Bcl2, androgen receptor (AR). Immunostaining was performed on a tissue microarray, including 40 prostates from fetuses aged 13-42 weeks and normal prostate tissue from 10 adults. In both solid buds and the basal compartment of canalized glands, cells expressed p63, CK5/6, CK19, CK18, BCL2, EGFR and were p27 negative. Luminal cells of fetal canalized glands continue to express CK19, EGFR, and BCL2, without p27 expression. In contrast, adult epithelial luminal cells showed diffuse AR and p27 expression, without CK19, BCL2, and EGFR staining. Proliferation was high and diffuse in fetal glands and rare and restricted to basal cells in adult glands. These results indicate that most fetal epithelial prostatic cells exhibit the phenotype of TA cells, suggesting their regulatory function in prostate development.