Prognostic molecular markers with no impact on decision-making: the paradox of gliomas based on a prospective study.

This study assessed the prognostic value of several markers involved in gliomagenesis, and compared it with that of other clinical and imaging markers already used. Four-hundred and sixteen adult patients with newly diagnosed glioma were included over a 3-year period and tumour suppressor genes, oncogenes, MGMT and hTERT expressions, losses of heterozygosity, as well as relevant clinical and imaging information were recorded. This prospective study was based on all adult gliomas. Analyses were performed on patient groups selected according to World Health Organization histoprognostic criteria and on the entire cohort. The endpoint was overall survival, estimated by the Kaplan-Meier method. Univariate analysis was followed by multivariate analysis according to a Cox model. p14(ARF), p16(INK4A) and PTEN expressions, and 10p 10q23, 10q26 and 13q LOH for the entire cohort, hTERT expression for high-grade tumours, EGFR for glioblastomas, 10q26 LOH for grade III tumours and anaplastic oligodendrogliomas were found to be correlated with overall survival on univariate analysis and age and grade on multivariate analysis only. This study confirms the prognostic value of several markers. However, the scattering of the values explained by tumour heterogeneity prevents their use in individual decision-making.

The status of CDKN2A alpha (p16INK4A) and beta (p14ARF) transcripts in thyroid tumour progression.

CDKN2A locus on chromosome 9p21 encodes two tumour suppressor proteins pl6INK4A, which is a regulator of the retinoblastoma (RB) protein, and p14ARF, which is involved in the ARF-Mdm2-p53 pathway. The aim of this study was to determine if CDKN2A gene products are implicated in differentiated thyroid carcinogenesis and progression. We used real-time quantitative RT-PCR and immunohistochemistry to assess both transcripts and proteins levels in 60 tumours specimens. Overexpression of p14ARF and pl6INK4A was observed in follicular adenomas, follicular carcinomas and papillary carcinomas, while downregulation was found in oncocytic adenomas compared to nontumoral paired thyroid tissues. These deregulations were statistically significant for pl6INK4a (P=0.006) in follicular adenomas and close to statistical significance for p14ARF in follicular adenomas (P=0.06) and in papillary carcinomas (P=0.05). In all histological types, except papillary carcinomas, we observed a statistically significant relationship between p14ARF and E2F1 (r=0.64 to 1, P<0.05). Our data are consistent with involvement of CDKN2A transcript upregulation in thyroid follicular tumorigenesis as an early event. However, these deregulations do not appear to be correlated to the clinical outcome and they could not be used as potential prognostic markers.

Prognostic value of increase in transcript levels of Tp73 DeltaEx2-3 isoforms in low-grade glioma patients.

Glial tumours are a devastating, poorly understood condition carrying a gloomy prognosis for which clinicians sorely lack reliable predictive parameters facilitating a sound treatment strategy. Tp73, a p53 family member, expresses two main classes of isoforms--transactivatory activity (TA)p73 and DeltaTAp73--exhibiting tumour suppressor gene and oncogene properties, respectively. The authors examined their expression status in high- and low-grade adult gliomas. Isoform-specific real-time reverse transcription-polymerase chain reaction was used for the analysis of Tp73 isoform transcript expression in a series of 51 adult patients harbouring glial tumours, in order to compare tumour grades with each other, and with non-tumoural samples obtained from epileptic patients as well. Our data demonstrate increase of TAp73 and DeltaTAp73 transcript levels at onset and early stage of the disease. We also show that DeltaEx2-3 isoform expression in low-grade tumours anticipates clinical and imaging progression to higher grades, and correlates to the patients' survival. Expression levels of P1 promoter generated Tp73 isoforms--and particularly DeltaEx2-3--indeed allow for prediction of the clinical progression of low-grade gliomas in adults. Our data are the first such molecular biology report regarding low-grade tumours and as such should be of help for sound decision-making.

Expression of TAp73 and DeltaNp73 isoform transcripts in thyroid tumours.

AIM: This study was aimed to determine p73 status in thyroid tumours. METHODS: Differential expression of the TAp73, DeltaTAp73 transcripts was measured in a panel of 60 thyroid malignancies by quantitative RT-PCR. RESULTS: By comparison to normal thyroid tissue surrounding the tumours, we observed significant downregulation of TP73 transcripts in adenomas and in differentiated carcinomas. Correlations were found in normal tissue specimens between the expression of TAp73 and DeltaNp73 transcripts and that of p53, p14ARF p16INK4a, but these correlations were lost in carcinomas (PTC or FTC). CONCLUSIONS: We have found significant variations of TAp73, DeltaNp73, p53, p14ARF p16INK4a, expressions and correlations between the expressions of those different genes in thyroid cancer.

[Gene MGMT and oligodendrogliomas. Fundamental data and short review of the literature]. / Gène MGMT et oligodendrogliomes. Données fondamentales et brève revue de la littérature.

MGMT (O6 methyl guanine methyl transferase) is a gene involved in DNA repair. Its mechanism of action is to remove alkyl groups created by alkylating chemotherapy and therefore induces chemoresistances. Recent studies show that this gene expression seems to be related to the promoter's methylation, which could predict a possible chemosensitivity. The study of MGMT could be of some therapeutic and prognostic interest. Few series of oligodendrogliomas have been published and their results appear to be controversial. This is probably due to both tumour heterogeneity and multiple parameters associated with chemosensitivity. To date, it thus appears difficult to choose the adjuvant treatment according to the sole status of MGMT.

Correlation of clinical features and methylation status of MGMT gene promoter in glioblastomas.

In an effort to extend the potential relationship between the methylation status of MGMT promoter and response to CENU therapy, we examined the methylation status of MGMT promoter in 44 patients with glioblastomas. Tumor specimens were obtained during surgery before adjuvant treatment, frozen and stored at -80 degrees C until for DNA extraction process. DNA methylation patterns in the CpG island of the MGMT gene were determined in every tumor by methylation specific PCR (MSP). These results were then related to overall survival and response to alkylating agents using statistical analysis. Methylation of the MGMT promoter was detected in 68% of tumors, and 96.7% of methylated tumors exhibited also an unmethylated status. There was no relationship between the methylation status of the MGMT promoter and overall survival and response to alkylating agents. Our observations do not lead us to consider promoter methylation of MGMT gene as a prognostic factor of responsiveness to alkylating agents in glioblastomas.

Topoisomerase II alpha and telomerase expression in papillary thyroid carcinomas.

BACKGROUND: Altered topoisomerase II alpha (Topo II alpha) expression and telomerase activity (TA) reflect tumour cell growth and malignant transformation. METHODS: We examined TA by using a TRAP assay and expression of Topo II alpha by immunohistochemical analysis in a series of 27 cases of papillary thyroid carcinoma (PTC). RESULTS: Topo II alpha labelling index (LI) ranged from 0.1 to 4.2% and was significantly associated with patient age (r=-0.42, p=0.003), with higher levels of Topo II alpha in patients under 40 years. There was no relationship between Topo II alpha LI, AGES score or other clinical outcome. TA was detected in 14 PTC, with relative levels ranging from 1.2 to 102 units. A significant positive correlation between the multiplicity of tumoral foci and the TA levels (p<10(-2)) was noted. CONCLUSION: We concluded that Topo II alpha cannot be used as a marker of tumour aggressiveness. Furthermore, enhanced Topo II alpha expression in PTCs from patients less than 40 years old suggests that this age group might benefit from Topo II inhibitor chemotherapy.

[Clearing up the p16INK4a-p14/p19ARF imbroglio?]. / L'imbroglio p16INK4a-p14/p19ARF: un dénouement en vue?

Since its discovery, the CDKN2/MTS1 locus has been considered as an important site for the understanding of cell cycle deregulations that are involved in cancer cell generation. A comprehensive approach of the respective roles played by the two p16INK4a and p14/p19ARF (ARF) proteins encoded by this locus was not yet achieved because of the structural intrication of their genes. Inactivation of the only p16INK4a gene in mouse allowed to get better insight into this puzzle. In vivo results presented by de Pinho's group showed that inactivation of both p16INK4a alleles generated a panel of various types of tumors from the 28th week following birth. Bern's group dit not confirm this result but showed that the presence of only one ARF functional copy increases sensitivity of p16-/- mice to tumor occurrence indicating that insufficient dosage of ARF protein may facilitate tumorigenesis. It seems now established that, at least in mouse, ARF controls senescence in vitro, immortalisation and transformation by oncogenic ras. p16INK4a inactivation appears to be crucial for the induction of carcinogens-induced tumors.

Human LPP gene is fused to MLL in a secondary acute leukemia with a t(3;11) (q28;q23).

The mixed lineage leukemia, MLL, gene is frequently rearranged in patients with secondary leukemia following treatment with DNA topoisomerase II inhibitors. By FISH and Southern blot analyses we identified a rearrangement in the MLL gene due to a novel t(3;11)(q28;q23) chromosomal translocation in a patient who developed AML-M5 3 years after treatment for a follicular lymphoma. Through inverse PCR, the LPP (lipoma preferred partner) gene on 3q28 was identified as the MLL fusion partner. LPP contains substantial identity to the focal adhesion protein, zyxin, and is frequently fused to HMGIC in lipomas. The breakpoint occurred in intron 8 of MLL and LPP. Two in-frame MLL-LPP transcripts, which fuse MLL exon 8 to LPP exon 9, were detected by RT-PCR, although the smaller of these contained a deletion of 120 bp from the MLL sequence. The predicted MLL-LPP fusion protein includes the A/T hook motifs and methyltransferase domain of MLL joined to the two last LIM domains of LPP. A reciprocal LPP-MLL transcript, predicted to include the proline-rich and leucine zipper motifs, and the first LIM domain of LPP were also detected by RT-PCR. In summary, LPP is a newly identified MLL fusion partner in secondary leukemia resulting from topoisomerase inhibitors. The MLL-LPP and LPP-MLL predicted proteins contain many of the features present in other MLL rearrangements.

Human ARF protein interacts with topoisomerase I and stimulates its activity.

The ARF gene (p19(ARF) in mouse and p14(ARF) in man) has become a central actor of the cell cycle regulation process as it participates to the ARF-MDM2-p53 pathway and the Rb-E2F-1 pathway. By use of immunoprecipitation and Western blotting (IP/WB), we now show that ARF physically associates with topoisomerase I (Topo I). ARF-Topo I immune complexes were detected in SF9 insect cells infected with recombinant baculoviruses encoding the two genes as well as in 293 cells that express endogenously these proteins. Preparations of a GST-ARF recombinant protein stimulated the DNA relaxation activity of Topo I but, in contrast, had no effect on the decatenation activity of Topo II. The Topo I stimulation was also detected in cell extracts of SF9 cells expressing both proteins. A confocal microscopy study indicated that part of ARF and Topo I colocalized in the granular component structure of the nucleolus. As a whole, our data indicate that Topo I is a new partner of ARF and suggest that ARF is involved in cell reactions that require Topo I.

Human ARF binds E2F1 and inhibits its transcriptional activity.

The INK4a/ARF locus which is frequently inactivated in human tumours encodes two different tumour suppressive proteins, p16(INK4a) and ARF. p16(INK4a) is a major component of the RB pathway. ARF is part of an ARF-mdm2-p53 network that exerts a negative control on hyperproliferative signals emanating from oncogenic stimuli. Among these is the transcription factor E2F1, a final effector of the RB pathway, that induces ARF expression. Recent data suggest that ARF function is not restricted to the p53 pathway. However, ARF target(s) implicated in this p53-independent function remains to be identified. We show that ARF is able to inhibit the proliferation of human cell lines independently of their p53 status. In this context, we demonstrate that ARF interacts physically with E2F1 and inhibits its transcriptional activity. Moreover, we show that mdm2 is required for the modulation of E2F1 activity by ARF. Beside the well-known p53 and mdm2 partners, these results identify E2F1 as a new ARF target. Thus, ARF can be viewed as a dual-acting tumour suppressor protein in both the p53 and RB pathways, further emphasizing its role in tumour surveillance.

Different combinations of genetic/epigenetic alterations inactivate the p53 and pRb pathways in invasive human bladder cancers.

Inactivation of both the pRb (pRb-cyclin D1/cyclin-dependent kinase 4/6-p16) and p53 (p53-p21(WAF1)-p14(ARF)) pathways is thought to be essential for immortalization in vitro and malignant transformation in vivo. We identified different combinations of pRb and p53 pathway alterations in 12 invasive transitional cell carcinomas (TCCs) and addressed the functional significance of the different combinations observed. Results showed four combinations of alterations including -pRb/-p53 (ie., pRb inactivated in the pRb pathway and p53 inactivated in the p53 pathway; four TCCs), -p16/-p53 (four TCCs), -p16/-p21(WAF1) (one TCC), and -p16/ -p14(ARF) (two TCCs). These groups include two new combinations (ie., -p16/-p53 and -p16/-p21(WAF1)) not reported previously for TCCs. An alteration in the key components of the p53 pathway was not detected in one invasive TCC that had inactivated p16. Note that all four TCCs with inactivated pRb had mutant p53; thus, the combinations of -pRb/ -p21(WAF1) and -pRb/-p14(ARF) were not observed. Only two of eight TCCs with altered p16 had concomitant p14(ARF) loss, demonstrating that simultaneous inactivation of these two 9p21INK4a tumor suppressor genes is not obligatory. To determine the biological phenotypes of TCCs with different combinations of pRb and p53 pathway alterations, their downstream responses to gamma radiation were studied in vitro. As expected, none of eight TCCs with mutant p53 responded to gamma radiation by elevation of p53, p21(WAF1), or mdm2 or by cell cycle arrest. Only two of four TCCs with wild-type p53 and wild-type pRb (the combination of -p16/-p14(ARF)) showed normal downstream responses to gamma radiation and underwent cell cycle arrest. Two TCCs with wild-type pRb and wild-type p53 (the combination of -pl6/-p21(WAF1) and one TCC with -p16) failed to show cell cycle arrest in response to radiation. This was attributed to the absence of p21(WAF1) in one TCC. In summary, these data support a model of invasive bladder cancer pathogenesis in which both the pRb and p53 pathways are usually inactivated and the biology of the tumor is impacted by the mechanism of their inactivations.

[The ARF-p53 pathway: a line of defence against oncogenic signals]. / La voie ARF-p53: une ligne de défense contre des signaux oncogéniques.

The MTS1 (Multiple Tumor Suppressor 1) locus is a very original one as its organization results in the expression of two alternative transcripts that encode two structurally and functionally different proteins: INK4a and ARF (also designated p19ARF in mouse and p14ARF in man). Recent findings indicate that the latter is a major component of a regulatory pathway of oncogenic signals culminating in p53 activation by stabilisation of the protein. While the importance of this pathway has been overtly established in animal experimental oncology, it still has to be further documented in human oncology in order for this gene to acquire its full biological significance.