Wilms’ tumor gene 1 (WT1) functions including some contradictory effects may be explained by the presence and interactions of its isoforms, however, their evaluation has been so far complicated by several technical problems. We designed unique quantitative PCR systems for direct quantification of the major WT1 isoforms A[EX5−/KTS−], B[+/−], C[−/+] and D[+/+] and verified their sensitivity, specificity and reproducibility in extensive testing. With this method we evaluated WT1 total and isoform expression in 23 normal bone marrow (BM) samples, 73 childhood acute myeloid leukemia (AML), 20 childhood myelodysplastic syndrome (MDS), 9 childhood severe aplastic anemia (SAA), 30 adult AML and 29 adult MDS patients. WT1 isoform patterns showed differences among these samples and clustered them into groups representing the specific diagnoses (P<0.0001). Isoform profiles were independent of total WT1 expression and possess certain common features—overexpression of isoform D and EX5[+] variants. The KTS[+]/KTS[−] ratio was less variable than the EX5[+]/EX5[−] ratio and differed between children and adults (P<0.001); the EX5[+]/EX5[−] ratio varied between diagnoses (AML vs MDS, P<0.001). These findings bring new insights into WT1 isoform function and suggest that the ratio of WT1 isoforms, particularly EX5 variants, is probably crucial for the process of malignant transformation.

BACKGROUND: Second cancers are an important cause of mortality and morbidity in long‐term survivors of testicular germ cell tumors (TGCTs). Studies on the impact of follow‐up for the first tumor on the outcome of second malignancies are lacking. The aim of this study was to study the details of diagnosis of second cancers and the role of focused oncology follow‐up.METHODS: Medical records and the electronic database of a tertiary referral center for germ cell neoplasms were searched for second cancers diagnosed in TGCT survivors. In a database of 1057 patients, 63 cases of metachronous second malignancies (26 contralateral testicular cancers and 37 nontesticular cancers) were found in 57 patients. Long‐term oncology follow‐up consisted of yearly history, physical examination, germ cell tumor markers, and imaging including abdominal computed tomography (CT) scans and chest x‐ray.RESULTS: The second malignancies occurred after a medium follow‐up of 9.9 years (range, 1.1‐33 years) after the diagnosis of the first tumor. Only 17 (27%) of the 63 second tumors were detected by oncology follow‐up investigations, and a further 12 (29%) were detected by nononcology physicians during a preplanned clinical visit. In 34 (54%) cases, patients themselves or their relatives initiated a clinical appointment because of symptoms. Follow‐up investigations all had low yields for the detection of second malignancies, although CT imaging did detect several cases of cancer at an early stage.CONCLUSIONS: In this retrospective study, most second cancers occurring in long‐term TGCT survivors were missed by regular oncology follow‐up that included yearly physical examination, tumor marker, and imaging. Cancer 2011;. © 2011 American Cancer Society.