Low WT1 Expression Research Articles

WT1 Expression as Follow up Marker in CML: Low Impact and Sensitivity Compared to BCR-ABL Specific RQ-PCR but High Impact for Detection of New Aberrant Clones in BCR-ABL Negative Follow up Samples

In CML real time PCR for the detection of residual disease usually has been done by the detection of the BCR-ABL-fusion transcript. In addition some reports have shown the usefulness of WT1 as a target for follow up in CML blast crisis. The aim of this study was to evaluate whether WT1 in addition to BCR-ABL assessment in imatinib treated CML can give any new information. Expression of BCR-ABL as well as WT1 (ELN assay) was normalized to ABL and expressed as % to ABL. In total 268 bone marrow samples (spls) of 40 patients (pts) have been analysed. Sixteen spls were newly diagnosed untreated CML. 252 spls were analysed at several timepoint during treatment with imatinib starting with a “near to untreated” sample with a >50% BCR-ABL/ABL ratio. According to standard definitions 6 of the 40 pts showed no response, 16 minor (MR), and 18 major molecular response (MMR) as determined by BCR-ABL expression. Three of the MR and one of the MMR pts relapsed. The 14 pretreatment spls showed a median WT1 expression of 3.36 (range 0.176–14.9) whereas the median BCR-ABL level was 56.6 (range 11.4–149.2). Thus the BCR-ABL level was more than one log above the WT1 level and was more uniform within the cohort. This lower total expression and a background level of 0.04 (as estimated from 7 normal bone marrows) results in 2–3 log lower sensitivity of WT1 compared to BCR-ABL. The correlation of WT1 and BCR-ABL in the total cohort of 268 spls was low (r=0.259). Median WT1 expression in BCR-ABL negative spls (n=15) was 0.055 and thus correlates to that of normal spls. Regarding the total follow up in all 40 pts a good correlation of WT1 and BCR-ABL was found only in 9 cases, all of which had a WT1 expression >10 at the start of follow up. 7 pts had a WT1 expression between 1–7 with good correlation to BCR-ABL but restricted sensitivity. 24 pts had a WT1 expression of <1 and a BCR-ABL ratio >50 at start of follow up. In these pts there was no correlation of BCR-ABL and WT1 during follow up. In two of these 24 pts there was even a negative correlation with increasing WT1 levels during FU even though BCR-ABL remained undetectable. Cytogenetic investigation of these two pts revealed the development of Ph-negative aberrant clones (one with +8 and one with +11). This result raised the hypothesis that increasing WT1 expression in BCR-ABL negative CML during follow up may indicate the development of BCR-ABL negative clones. Therefore we measured the WT1 expression in a new cohort of 26 CML pts that developed Ph-clones during follow up. All these pts had low (<0.5) or undetectable BCR-ABL. Chromosomal aberrations in these cases were −7 (n=1), +8 (n=12), +11 (n=1), del(20q) (n=1), −Y (n=10)–X (n=1). A high WT1 expression (range 5–177) was detected in the two cases with +11 and −7 and in 10 of 12 cases with +8. A low WT1 expression (<0.5) was detected in the case with 20q- and in all 10 cases with–Y. An intermediate expression of 1.5 was detected in the case with +X. In summary a high WT1 expression was detected in 12/26 (46%) of all cases with new chromosomal aberrations in a Ph-negative clone and in 12/16 (75%) excluding the 10 cases with -Y. So far, the clinical relevance of Ph-negative clones is unclear. In rare cases the development of secondary MDS has been described. The cases that were followed here were all still in CR of CML with no signs of a secondary disease. However, high WT1 expression was detected in most cases with +8 but never with–Y, possibly indicating unequal biological relevance of these aberrations. The 10–100-times elevated WT1 expression in the +8 and +11 cases suggest that these aberrations may be of adverse impact. However, further clinical follow up is needed to show the outcome in these pts. In contrast, none of the cases with–Y revealed elevated WT1 expression suggestive for the expansion of healthy–Y clones that frequently can occur in a normal male bone marrow. 21 pts with Ph-negative clones could be followed for 13–36 months. There was a good correlation of the WT1 expression level with the number of metaphases carrying the chromosomal aberration (r=0.729). In one case an increase of WT1 expression was measured 11 months before detection of the evolving +8 suggestive of a preexisting cytogenetically undetected clone. In conclusion,BCR-ABL is superior to WT1 as marker for follow up in CML.Increasing WT1 levels in BCRABL negative follow up spls can indicate the development of Ph-negative clones under imatinib treatment.Future studies will show whether the WT1 level in Ph-clones may indicate the relevance of certain aberrations for outcome in these patients.

Real-Time Quantitative PCR (RQ-PCR) Detection of Minimal Residual Disease (MRD) by Optimized WT1 Assay To Enhance Risk Stratification in Acute Myeloid Leukemia (AML): A European LeukemiaNet Study.

Relapse remains the major cause of treatment failure in AML; however, RQ-PCR assays to detect leukemic fusion transcripts have been shown to identify reliably those patients at highest risk of relapse, allowing development of a more individualized treatment approach. In cases lacking a leukemia-specific MRD marker, quantification of genes over-expressed in AML e.g. Wilms' Tumor gene (WT1) could provide more a precise measurement of disease response and quality of remission, potentially enhancing risk scores such as the one developed by the MRC used to identify those patients most and least likely to benefit from allogeneic transplant in first CR. WT1 is over-expressed in at least 75% of AML cases. Within the European LeukemiaNet we systematically evaluated 9 published and “in house” WT1 assays. Assays were excluded due to demonstrated lack of RNA-specificity or location within the 3′ region of the gene which has been shown to be subject to deletion or mutation in AML. An assay located within the 5′ region associated with superior sensitivity was ultimately selected following parallel testing in 11 labs and evaluated in 238 diagnostic peripheral blood (PB) and 386 bone marrow (BM) samples. WT1 was over-expressed in the majority, with comparable levels in PB and BM (PB - median 4637 WT1 copies/104ABL copies, range 0–1132709; BM - median 7212, 0–750571), as compared to normal BM (median 19.8, 0–213), PB (median 0.01, 0.01–47.6) and PBSCs (median 6.1, 0–39). In cases over-expressing WT1, kinetics of transcript reduction were evaluated following induction. A greater response was associated with a significantly reduced risk of relapse (hazard ratio 0.65 per log reduction (95% CI 0.43–0.96), p=0.03), although this failed to remain significant when adjusted for age, presenting WBC and cytogenetic risk group, which are key variables in the MRC risk index. Indeed, there was a highly significant correlation between larger log reduction in normalized WT1 transcript level and better risk score (p=0.0001). Sequential analysis of PB and BM samples from 15 AML cases with low WT1 expression (<250 copies) showed no significant modulation in transcript level on regeneration after chemotherapy, indicating that in WT1+ AML, transcript levels detected in follow-up samples reliably reflect disease status. This study provides evidence that recognized pre-treatment risk factors for relapse correlate closely with kinetics of response to induction therapy and lend support to the evaluation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification and identify those patients most suited to proceed rapidly to allogeneic transplant.

Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic significance, correlation with disease status and residual disease detection by flow cytometry.

The clinical significance of WT1 gene expression at diagnosis and during therapy of AML has not yet been resolved. We analysed WT1 expression at presentation in an unselected group of 47 childhood AML patients using real-time quantitative reverse-transcription PCR. We also showed that within the first 30 h following aspiration RQ-RT-PCR results were not influenced by transportation time. We observed lower levels of WT1 transcript in AML M5 (P = 0.0015); no association was found between expression levels and sex, initial leukocyte count and karyotype-based prognostic groups. There was significant correlation between very low WT1 expression at presentation and excellent outcome (EFS P = 0.0014). Combined analysis of WT1 levels, three-colour flow cytometry residual disease detection and the course of the disease in 222 samples from 28 children with AML showed remarkable correlation. Fourteen patients expressed high WT1 levels at presentation. In eight of them, who suffered relapse or did not reach complete remission, dynamics of WT1 levels clearly correlated with the disease status and residual disease by flow cytometry. We conclude that very low WT1 levels at presentation represent a good prognostic factor and that RQ-RT-PCR-based analysis of WT1 expression is a promising and rapid approach for monitoring of MRD in approximately half of paediatric AML patients.

Immunohistochemical detection of the Wilms' tumor gene (WT1) in epithelial ovarian tumors.

The Wilms' tumor gene WT1 plays multiple roles in the development of the genitourinary organs and Wilms' tumors. The aims of this study were to immunohistochemically evaluate WT1 expression in normal female genital tissues and in epithelial ovarian tumors and to look for correlations between WT1 expression and histologic subtypes and cell proliferation in epithelial ovarian tumors. In normal female genital organs, WT1 expression was recognized in ovarian surface epithelium, the lining of inclusion cysts, and tubal epithelium, but not in the cervical or endometrial epithelium. In epithelial ovarian tumors, serous tumors generally revealed a high WT1 expression. Among adenocarcinomas, serous carcinoma revealed a significantly higher WT1 expression than the other histologic subtypes. There were no significant correlations between the WT1 labeling index and the Ki-67 labeling index, and no significant difference in survival between those showing high and low WT1 expression among the malignant cases. These results suggest that WT1 expression may be related to cell differentiation, and that the histologic subtypes of epithelial ovarian carcinomas may differ considerably in their biological characteristics.