Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories.

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

Predictive value of pretransplantation molecular minimal residual disease assessment by WT1 gene expression in FLT3-positive acute myeloid leukemia.

The FMS-like tyrosine kinase 3 (FLT3) mutation in acute myeloid leukemia (AML) is a negative prognostic factor and, in these cases, allogeneic stem cell transplantation (allo-SCT) can represent an important therapeutic option, especially if performed in complete remission (CR). However, it is increasingly clear that not all cytological CRs (cCRs) are the same and that minimal residual disease (MRD) before allo-SCT could have an impact on AML outcome. Unfortunately, FLT3, due its instability of expression, is still not considered a good molecular MRD marker. We analyzed the outcome of allo-SCT in a population of FLT3-positive AML patients according to molecular MRD at the pretransplantation workup, assessed by the quantitative expression evaluation of the panleukemic marker Wilms' tumor (WT1) gene. Sixty-two consecutive AML FLT3-positive patients received allo-SCT between 2005 and 2016 in our center. The median age at transplantation was 55 years. The quantitative analysis of the WT1 gene expression (bone marrow samples) was available in 54 out of 62 (87%) cases, both at diagnosis (100% overexpressing WT1 with a mean of 9747 ± 8064 copies) and before allo-SCT (33 WT1-negative and 21 WT1-positive cases at the pretransplantation workup). Of these cases, 33/54 (61%) were both in cCR and molecular remission (WT1-negative) at the time of transplantation, 13/54 (24%) were in cCR but not in molecular remission (WT1-positive), and 8/54 (15%) showed a cytological evidence of disease (relapsed or refractory). Both post-allo-SCT overall survival (OS) and disease-free survival (DFS) were significantly better in patients who were WT1-negative (WT1 <250 copies) at the time of transplantation compared with those who were WT1-positive (WT1 >250 copies), with a median OS and DFS not reached in the WT1-negative group and 10.2 and 5.5 months, respectively, in the WT1-positive group (OS log-rank p = 0.0005; hazard ratio [HR] = 3.7, 95% confidence interval [95% CI] = 1.5-9; DFS log-rank p = 0.0001; HR = 4.38, 95% CI = 1.9-10). Patients with cCR who were WT1-positive had the same negative outcome as those with a cytological evidence of disease. The relapse rate after allo-SCT was 9% (3/33) in pre-allo-SCT WT1-negative cases and 54% (7/13) in WT1-positive cases (p = 0.002). At multivariate analysis, WT1 negativity before allo-SCT and grade <2 acute graft versus host disease were the only independent prognostic factors for improved OS and DFS. These data show that pre-allo-SCT molecular MRD evaluation through WT1 expression is a powerful predictor of posttransplantation outcomes (OS, DFS, relapse rate). Patients with both cCR and a WT1-negative marker before allo-SCT have a very good outcome with very low relapse rate; conversely, patients with positive molecular MRD and refractory/relapsed patients have a negative outcome. The WT1 MRD stratification in FLT3-positive AML is a valuable tool with which to identify patients who are at high risk of relapse and that could be considered from post-allo-SCT prophylaxis with FLT3 inhibitors or other strategies (donor lymphocyte infusion, tapering of immunosuppression, azacitidine).

Quantitative assessment of WT1 gene expression after allogeneic stem cell transplantation is a useful tool for monitoring minimal residual disease in acute myeloid leukemia.

INTRODUCTION: WT1 overexpression is described in several oncological diseases including acute myeloid leukemia (AML). Quantification of WT1 in bone marrow samples may be useful as a marker of minimal residual disease (MRD) and may predict the relapse of AML after allogeneic hematopoietic stem cell transplant (HSCT). METHODS AND RESULTS: The quantitative expression of WT1 was measured in 38 AML patients (16 males and 22 females) at diagnosis, at the time of transplant and after the allogeneic HSCT (at precise time points). All cases showed high WT1 expression levels at diagnosis with a mean of 4189 (SD 3325) and a median of 3495 (range 454-13923) copies WT1/10(4)Abl. At transplant, 25 patients (66%) were in complete cytologic remission (CcR) and 13 (34%) had refractory or relapsed AML. Bone marrow samples from patients transplanted in CcR showed significantly lower WT1 expression levels during HSCT compared with the samples from patients with a relapsed or refractory AML (P = 0.004). After HSCT, a rapid decline in WT1 expression levels was observed in all patients who attained or maintained a condition of CcR. Six of 38 patients (13%) relapsed after HSCT and all of them had an increase in WT1 expression at/or before relapse. Five of these six patients died of leukemia and one was successfully reinduced with donor lymphocyte infusion (DLI) + chemotherapy with a rapid reduction of WT1 levels. Besides, we found a complete concordance between WT1 expression levels and other disease markers (when available). CONCLUSIONS: In our experience, there was a complete concordance between WT1 expression levels (measured by quantitative RT-PCR at precise time points) and status of AML before and after allogeneic HSCT. WT1 may be useful as a non-specific leukemia marker for monitoring MRD and as a predictor of AML clinical relapse. Based on these results, cases with increase of WT1 levels after HSCT and without graft vs. host disease may be candidate to discontinuation of immunosuppression and/or DLI therapy.