High WT1 Research Articles

Effects of different surgical extents on prognosis of patients with malignant ovarian sex cord-stromal tumors: a retrospective cohort study.

Malignant ovarian sex cord-stromal tumors are rare neoplasms that account for approximately 5-7% of all ovarian malignancies, and they are primarily treated with surgery. The prognosis of patients with different surgical extents remains controversial. Therefore, the effects of different surgical extents on the prognosis of patients were explored in this retrospective cohort study. Patients with malignant ovarian sex cord-stromal tumors who underwent surgical treatment from January 2000 to December 2019 were selected. Disease-freesurvival and overall survival rates werecalculatedbytheKaplan-Meier method andcompared by thelog-rank test. Prognosis factors were identified by Coxregression analysis. P < 0.05 was considered a statistically significant difference. A total of 278 patients with an average age at onset of 42 (8-78) years old were enrolled. The median follow-up time was 73months. There was no significant difference in disease-free survival and overall survival rates between patients who underwent fertility-sparing surgery and those who underwent Non-fertility-sparing surgery, and between patients underwent staging surgery and those underwent Non-staging surgery.Age < 40years (P = 0.024), stage II-III (P = 0.038), a high CA125 level (P = 0.035) and WT-1 (+) (P = 0.016) were independent risk factors for recurrence. In conclusion, different surgical extents have no significant influence on recurrence and survival status of patients with malignant ovarian sex cord-stromal tumors.

Role of WT1 in Measurable Residual Disease Follow-Up in the Post Allogeneic Stem Cell Transplant Setting.

Objectives: The Wilms' tumor gene 1 (WT1) plays a critical role in cell development and the regulation of essential genes involved in cell growth and metabolism. In the context of hematopoietic tumors, including acute myeloid leukemia (AML), WT1 has been identified as a potential marker for measurable residual disease (MRD) assessment. Relapse after allogeneic hematopoietic stem cell transplantation (allo-SCT) remains a significant challenge in AML treatment, highlighting the importance of MRD monitoring for risk stratification and treatment decisions. This study aimed to investigate the clinical significance of WT1 as a molecular marker for MRD and its correlation with chimerism in AML patients post-allo-SCT setting. Methods: We have included 58 patients with WT1-expression-positive acute myeloid leukemia (AML) who received allo-SCT in our center between 2016-2022. The exclusion criteria are as follows: not having WT1 polymerase chain reaction (PCR) measurement at diagnosis, not receiving allo-SCT, and not having a serial measurement of WT1 post-transplant. Pre- and post-transplant assessments were made with flow cytometry, WT1 PCR, and bone marrow morphological evaluations. Statistical analyses were carried out to explore correlations between WT1 levels, MRD markers, and chimerism post-transplantation. Results: We found that WT1 had a significant correlation with flow cytometry and bone marrow morphological evaluation, but not with chimerism. Interestingly, high WT1 expressors exhibited a more robust correlation with chimerism compared to the general cohort. The negative predictive value for post-allo-SCT relapse was 91.8% for the whole WT1 cohort; for high WT1 expressors, it was similar, at 87.5%. The negative predictive value for post-allo-SCT relapse was high for the whole WT1 cohort; for high WT1 expressors, it was similar. The WT1 MRD assay showed a high negative predictive value for post-allo-SCT relapse, consistent across both the entire cohort (91.8%) and high WT1 expressors (87.5%). Conclusions: WT1 expression levels may serve as a valuable ancillary marker in MRD assessment and relapse prediction post-allo-SCT in AML patients, particularly for those lacking specific fusion genes or mutations. However, further large-scale, controlled studies are needed to standardize WT1 MRD assays and establish clear guidelines for their clinical application.

Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.Graphical abstract

Clinical significance of WT1 in the evaluation of therapeutic effect and prognosis of non-M3 acute myeloid leukemia

ABSTRACT To explore the clinical significance and prognosis of acute myeloid leukemia (AML) patients with WT1 mutations.In total, the clinical data of 269 adult patients with non-M3 AML were considered retrospectively. From these patients, 153 carried WT1 mutation whereas 116 were negative. WT1 mutation positive patients were further divided into WT1 low expression and high expression groups base on the expression level of WT1 by qPCR at diagnosis (cut off: 170500). Survival and therapeutic effect analysis were performed for the above patients with different interfering factors such as co-mutations, the extent of WT1 log reduction and the chemotherapy regimens. Patients with high WT1 expression have higher rate of relapse. We can accurately identify patients with inferior outcomes when we take the following factors into consideration: the WT1 expression level at diagnosis; different prognostic factors including co-mutations (especially NPM1 and FLT3-ITD); the log reduction of WT1 after induction therapy and the risk of stratification. Idarubicin + Cytarabine (IA) regimen could reduce the expression level of WT1 after treatment, and Allo-HSCT played an important role in improving the prognosis of patients with WT1 high expression and patients with WT1 negativity. Among the relapsed patients, there existed a rising trend of WT1-MRD in advance than MFC-MRD and that of patients with continuous complete remission (CR). Different clinical background should be taken into consideration when we judge the prognosis and therapeutic effect of patients with WT1 mutations. In addition, WT1 may be an optional MRD marker, which needs regular monitoring.

High WT1 expression predicted induction chemotherapy failure in acute myeloid leukemia patients with non-favorable cytogenetic risk.

The prognostic significance of WT1 expression at diagnosis in acute myeloid leukemia (AML) remains obscure, and subgroup analysis is the way for clarification. We previously reported the results in t(8;21) AML. In this study, 437 consecutive adult AML patients with non-favorable cytogenetic risk were enrolled. All patients were tested WT1 transcript levels using real-time quantitative PCR at diagnosis; AML-related common fusion genes, KMT2A-PTD, FLT3-ITD, NPM1, CEBPA and TP53 mutations were simultaneously tested. 92.4% of patients overexpressed WT1 compared to normal bone marrow. The existence of FLT3-ITD, NPM1 mutation and the absence of CEBPA biallelic mutation were significantly related to higher WT1 expression. The cutoff value for WT1 was determined by performing receiver operating characteristic curve analysis in regard to complete remission (CR) achievement and was used to categorize patients into low-expression (WT1-L) and high-expression (WT1-H) groups. In the entire cohort, WT1-H was significantly associated with a lower 1-course and 2-course CR rate (P < 0.0010 and P = 0.0060) but was not related to relapse-free survival (RFS). Multivariate analysis showed that WT1-H was an independent adverse prognostic factor for both 1-course and 2-course CR achievement. Subgroup analysis was further performed. WT1-H had a significant adverse impact on CR achievement within intermediate-cytogenetic risk, high-cytogenetic risk, ELN-defined-intermediate-risk, normal karyotype, KMT2A rearrangement, FAB-M2, FAB-M5 and NPM1 mutation (+) subgroups, whereas it had no impact within ELN-defined-low-risk, ELN-defined-high-risk, FAB-M4, FLT3-ITD mutation (+) and CEBPA biallelic mutation (+) subgroups. Moreover, WT1-H patients had a significantly lower RFS rate than WT1-L patients within both FAB-M5 and KMT2A rearrangement subgroups (P = 0.010 and 0.028), whereas WT1 had no impact on RFS within other subgroups mentioned above (all P > 0.05). Therefore, high WT1 expression at diagnosis independently predicted induction chemotherapy failure in AML patients with non-favorable cytogenetic risk, and it was related to relapse just within FAB-M5 and KMT2A rearrangement subgroups.

Bone Marrow WT1 mRNA Upregulation in Acute Myeloid Leukemia (AML) in Complete Molecular Remission (mCR): Understanding the Limitations and Advantages of WT1 As a Measurable Residual Disease (MRD) Target

Introduction WT1 mRNA levels assessed by q-PCR have been employed as a marker of remission in AML patients undergoing intensive chemotherapy (Nomdedeu J. et al. Leukemia 2013;27:2157) and hematopoietic cell transplantation (HCT) (Nomdedeu J et al. Biol Blood Marrow Transplant 2018;24:55). Patients with adjusted bone marrow WT1 mRNA levels greater than 100 copies showed a high probability of relapse. Despite the usefulness of WT1 quantification in this and other settings, this marker is scarcely employed as an MRD target because levels greater than >100 copies can also be observed in normal bone marrows. Furthermore, 10-20% of AML at diagnosis and most chronic myelomonocytic leukemia (CMML) cases lack WT1 upregulation. The high levels of bone marrow WT1 copy numbers observed in most AML, decline in responding patients, although the utility, if any, of WT1 levels in long-term survivors of AML is currently unknown. We investigated the frequency and biological findings of mCR AML samples showing WT1 upregulation(>100 copies) compared to that mCR AML with concordant low WT1 levels. Patients and methods The study included 717 bone marrow samples from 80 AML patients (23 patients with RUNX1-RUNX1T1, 18 pts with CBFb-MYH11, four pts with NPM1 mutations, and 35 pts with PML-RARa). Samples were obtained at diagnosis (58 samples) and the follow-up (659 samples, all in mCR and without relapses) from one month to 219 months after diagnosis. Chimeric transcripts and WT1 levels were investigated by standardized real-time methods (ELN). Hemoglobin and leukocyte counts were also recorded. Flow cytometry immunophenotyping was also performed using four-color flow cytometry. We analyzed the percentage of CD34+, CD117+, CD123+ cells, and the Myeloid/Lymphoid CD34 ratio. nCounter Stem cell panel (Nanostring) was used to investigate the differences between that AML in mCR, showing high (>100) and low (<100) WT1 levels. In an additional cohort (11 samples: 4 with WT1>100 and 7 with WT1 <100), we performed bulk RNAseq (Novogene) to investigate enriched genes in samples with high WT1 levels. Results Thirty-five patients had at least one sample with WT1 levels greater than 100 copies (44%) despite being in mCR. Twelve samples had WT1 levels greater than 300 copies. In one case, this level reached 770 copies. These WT1 "peak" samples were distributed as follows: 49/306 PML-RARa, 25/161 RUNX1-RUNX1T1, 10/174 CBFb-MYH11, and 1/18 NPM1. Patients showing WT1 peaks were older than those without elevations (47 years vs. 36 years, p:0.003) For PML-RARa samples, "peaking" was associated with low levels of hemoglobin (133 g/L for <100 copies vs. 125 g/L >100 copies, p:0.009). For RUNX1-RUNX1T1, "peaking" was linked to less platelets (132x109 vs. 197x109 p:0.05) , higher CD34 (1.5% vs. 1% p:0.05) and also higher CD117+ cells (1.6 vs.1%, p:0.01). Lastly, CBFb-MYH11+ samples with WT1 peaks had higher leukocyte counts that those showing low WT1 levels (4.5x109 vs. 2.9x109, p:0.006). Nanostring comparisons between mCR WT1 high vs. low samples showed upregulation of TOR1A, PIK3CB, PTPN6, and PGM1 with downregulation of HMGA1, HOXB2 and CDC6. RNA seq analysis detected an enrichment of SUZ12 targets in those samples with high WT1 levels. Conclusions Bone marrow mRNA WT1 levels commonly exceed the clinical relevant value of 100 copies in AML samples in mCR (13%). Peak samples were related to peripheral blood values and/or raised numbers of immature bone marrow cells (CD34, CD117). Bone marrow WT1 although unable to accurately predict relapses in long-term survivors of AML may be a non-invasive sensor of a yet undefined myeloid state.

CIC-rearranged sarcoma with rhabdoid features: a clinicopathological analysis

Objective: To investigate the histopathologic, immunohistochemical, molecular genetic characteristics of CIC-rearranged sarcoma (CRS) with rhabdoid features. Methods: The clinical and pathologic data of two cases of CRS diagnosed between 2019 and 2021 at the Department of Pathology, Jiangsu Province Hospital were analyzed. Immunohistochemical study and fluorescence in situ hybridization (FISH) were performed. The relevant literature was reviewed. Results: Both patients were female, one was 58 years old, with tumor located in left thigh; the other was 43 years old, with tumor located in left pelvic cavity. Microscopically, both tumors were composed of small to medium-sized round, oval cells, arranged in nodules or sheets. The tumor cells showed irregular nuclear outline, coarse chromatin with prominent nucleoli and brisk mitotic activity. Both cases showed rhabdoid phenotype with myxoid stromal changes. Immunohistochemically, both cases were positive for CD99 and c-myc. High WT1 reactivity was seen in classic area, with low reactivity in rhabdoid area. There was no INI1 lost in both cases. Both were negative for NKX2.2 and NKX3.1. By FISH both cases demonstrated convincing break-apart signals of CIC gene. One patient died of disease after 1 month, and the other died of disease after 3 months. Conclusions: CRS is a small round cell undifferentiated sarcoma of the bone and soft tissue defined by molecular genetic characteristics, and may show atypical morphologic and immunophenotypic characteristics such as rhabdoid features. A correct understanding of its rare morphologic and immunophenotypic characteristics, combined with molecular pathologic detection, is conducive to correct diagnosis.

PB2039: CLINICAL FEATURES AND NEXT-GENERATION SEQUENCING LANDSCAPE OF ESSENTIAL THROM-BOCYTHEMIA，PREFIBROTIC PRIMARY MYELOFIBROSIS, AND OVERT FIBROTIC PRIMARY MYELOFIBROSIS: A CHINESE RETROSPECTIVE STUDY

Background: Since prefibrotic primary myelofibrosis (pre-PMF) was recognized as a separate enti-ty in the 2016 revised classification of MPN differed from essential thrombocythemia (ET) or overt fibrotic primary myelofibrosis (overt PMF), it has been a subject of de-bate among experts due to its indefinite diagnosis. Aims: We conducted a retrospective study of patients with myeloproliferative neoplasms (MPNs) in China, including 56 ET pa-tients, 19 pre-PMF patients, and 43 overt PMF patients. Methods: We conducted a retrospective study of patients with BCR-ABL-negative MPN from October 2014 to May 2021 in the Fourth Affiliated Hospital of Zhejiang University School of Medicine. Patients who were diagnosed with ET, pre-PMF or overt PMF according to the 2016 WHO revised Classification of MPN were included. Patients with incomplete information from laboratory tests and medical records were excluded. This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Zhejiang University School of Medicine in line with the principles of the Declaration of Helsinki.Statistical analyses were performed using SPSS statistics 26.0 and GraphPad Prism 8.0 software. Results: Pre-PMF patients exhibited higher leukocyte counts[14.2(6.0-28.1)×109/L vs 9.6(4.0-55.0)×109/L, P=0.003], LDH values[307(233-479)U/L vs 241(129-1182)U/L, P<0.001], onset ages[67(32-76)years vs 50(16-79) years, P=0.006], a higher frequen-cy of splenomegaly(47.4% vs 16.7%, P=0.018) and hypertension(57.9% vs 23.2%, P=0.005) than ET patients. On the other hand, pre-PMF patients had higher platelet counts[960(500-2245)×109/L vs 633(102-1720)×109/L, P=0.017], haemoglobin levels[152(115-174)g/L vs 119(71-200)g/L, P=0.003], lower LDH val-ues[307(233-479)U/L vs 439(134-8100)U/L, P=0.007] and a lower frequency of splenomegaly(47.4% vs 75.6%, P=0.031) than overt PMF patients. Next-generation sequencing landscape was performed in 50 patients, revealed the frequency of EP300 mutations was significantly increased in pre-PMF patients compared with ET and overt PMF patients (60% vs 10% vs 15.79%, P=0.033), and WT1 was more often overexpressed (WT1/ABL1 copies≥1.0%) in patients with overt PMF than in those with ET or pre-PMF(54.55% vs 16.67% vs 17.65%, P=0.009). In terms of outcome, male sex, along with symptoms including MPN10, anaemia (haemoglobin<120g/L), thrombocytopenia (platelet count<100×109/L), leucocytosis (leukocyte counts>13×109/L), high LDH value (>350U/L), splenomegaly, WT1 overexpres-sion(WT1/ABL1 copies≥1.0%), KMT2A, ASXL1 and TP53 mutations, indicated a poor prognosis for PMF patients. The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011; the 2019–2024 Academician Workstation of the Fourth Affiliated Hospi-tal of the Zhejiang University School of Medicine; the 2019–2022 Key Medical Dis-cipline (Hematology) Fund of Jinhua, China. *Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. E-mail: [email protected] Image:Summary/Conclusion: In summary, the results of this study indicated that comprehensive evaluation of BM features, clinical phenotypes, haematologic parame-ters, and molecular profiles is needed for the accurate diagnosis and treatment of ET, pre-PMF, and overt PMF patients.

P790: THE EXPRESSION LEVEL OF WT1 MRNA IN PATIENTS WITH IDIOPATHIC CYTOPENIA OF UNDETERMINED SIGNIFICANCE (ICUS) PROGRESSING TO MYELODYSPLASTIC SYNDROME (MDS)

Background: Idiopathic cytopenia of undetermined significance (ICUS) is defined as patients with one or more unexplained cytopenia who do not meet diagnostic criteria for myelodysplastic syndrome (MDS) or another hematologic disorder. In the clinical course of ICUS, some patients eventually have typical MDS changes. Until now, there is no effective method to predict the development of the disease. Recent studies have shown that the expression level of WT1 mRNA in peripheral blood has important clinical significance in the diagnosis, occurrence, development and prognosis of MDS. At the same time, some studies have proved that there is a significant difference in the expression level of WT1 mRNA between MDS and hemocytopenia caused by other reasons. Aims: In order to clarify the clinical significance of WT1 mRNA expression in the progression of ICUS to MDS. Methods: Between April 2017 and January 2020, untreated adult patients with ICUS or lower-risk MDS were enrolled in the study. All patients detected the WT1 mRNA expression of bone marrow and peripheral blood at diagnosis. Then patients with ICUS were followed up. Every three months, the patients with ICUS detected the WT1 mRNA expression of peripheral blood, until the patient progressed to MDS. The results of WT1 transcript levels are depicted as WT1 (copies/mL)/c-ABL (copies/mL) as a percentage. Results: Total of 80 patients with ICUS and 40 patients with lower-risk MDS were enrolled. At diagnosis, the median expression level of WT1 mRNA in MDS group was 5.99 (0.08-79.27), and that in ICUS group was 0.50 (0.16-22.48). There was significant difference between them (P = 0.000). Then 80 patients with ICUS were followed up to January 2022. The median follow-up time was 30 months (11-54 months). 8 patients developed MDS, the progression rate was 10%, and the median progression time was 6.5 months (2-19 months). The median expression level of WT1 mRNA at the initial diagnosis in ICUS group without progressing to MDS was 0.44 (0.16-7.58), and that in progressing to MDS group was 9.44 (0.26-22.48). There was significant difference between the two groups (P = 0.031). The median expression level of WT1 mRNA was 19.185 (0.73-42.30) when MDS was diagnosed. The median maximum expression level of WT1 mRNA in peripheral blood of ICUS group without MDS was 0.98 (0.22-7.58). There was significant difference between the two groups (P = 0.007). Summary/Conclusion: The study clarified the WT1 mRNA expression status of ICUS patients. There were significant differences in WT1 mRNA expression level between patients with ICUS and lower-risk MDS, and the WT1 mRNA expression level of patients with lower-risk MDS was relatively high. The study confirmed that patients with high WT1 mRNA expression level in the patients with ICUS (WT1 mRNA expression level > 9.44) are very likely to progressing to MDS; Dynamic monitoring of WT1 mRNA expression in peripheral blood is an effective method for following-up after ICUS diagnosis. The progressive increase of WT1 mRNA expression is one of the indicators for researchers to judge the progress of ICUS to MDS.

PB1895: ANALYSIS REVEALS CANCER-ASSOCIATED GENE MUTATIONS IN CHRONIC MYELOID LEUKEMIA PATIENTS ：A CHINESE MONOCENTRIC RETROSPECTIVE STUDY

Background: Recent genetic studies have shown that tyrosine kinase inhibitors (TKI) resistant or intolerant Chronic myeloid leukemia (CML) patients often carry one or more genetic changes related to myeloid malignancies, suggesting that additional mutations other than ABL1 mutation may lead to TKI treatment failure or disease progression. Aims: We performed sequencing of 22 CML patients using next-generation sequencing (NGS). 23 CML patients tested WT1 expression detection. Methods: The second cohort (No. 1) tested by second-generation sequencing included 22 CML patients. The second cohort (No. 2) tested by WT1 expression detection included 23 CML patients. The study has been approved by the institutional ethics committee and patients provided written informed consent in accordance with the Fourth Affiliated Hospital of Zhejiang University School of Medicine.Definitions Response was defined as BCR-ABL1 transcript levels according to the International Scale (IS) ≤10%, ≤1% and ≤0.1% at 3, 6 and 12 months after initiating TKI-therapy. Molecular response with 4.0 log reduction (MR4.0) was defined as 0.01%. Molecular response with 4.5 log reduction (MR4.5) was defined as 0.0032% or less BCR-ABLtranscript level. The comparison between groups of measurement data showed that the data with normal distribution were analyzed by one-way ANOVA or t-test. Results: Overall, a total of 51 mutations involving 25 genes were detected most frequently in the ASXL1 (40.9%), KMT2C (27.3%), DIS3 (18.2%), ATM (13.6%), DNMT3A (13.6%) and NOTCH3 (13.6%) genes. NOTCH3 and RELN mutations were only 3/20 (10%) in those who did not reach 12M-MMR group. Although the difference was not statistically significant (P = 0.214, P = 0.476). The number of median mutant genes in reaching MR4.5 group was 1 (0-3). The number of median mutant genes in not reaching MR4.5 group was 4 (0-6), and the difference was statistically significant (P = 0.033). ASXL1 mutation in reaching MR4.0 group and non reaching MR4.0 group was statistically significant (P = 0.023). With KMT2C mutation group had lower PFS (P = 0.079). There was a potential statistical difference between low WT1 expression group and high WT1 expression group in early EMR after a 3-month treatment (P = 0.089).The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011; the 2019–2024 Academician Workstation of the Fourth Affiliated Hospi-tal of the Zhejiang University School of Medicine; the 2019–2022 Key Medical Dis-cipline (Hematology) Fund of Jinhua, China. *Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China.E-mail: [email protected] Image:Summary/Conclusion: Genomic risk assessment can improve the risk stratification of clinical diagnosis, and can more accurately identify patients with poor effect of TKI treatment in the era of TKI treatment. The treatment effect of CML patients with NOTCH3, RELN and ASXL1 mutations and more than two gene mutations is poor. CML patients with high expression of WT1 at diagnosis have poor therapeutic effect in the early stage.

Clinical features and next-generation sequencing landscape of essential thrombocythemia, prefibrotic primary myelofibrosis, and overt fibrotic primary myelofibrosis: a Chinese monocentric retrospective study.

Since prefibrotic primary myelofibrosis (pre-PMF) was recognized as a separate entity in the 2016 revised classification of MPN differed from essential thrombocythemia (ET) or overt fibrotic primary myelofibrosis (overt PMF), it has been a subject of debate among experts due to its indefinite diagnosis. We retrospectively reviewed the clinical parameters, haematologic information, and genetic mutations of patients who were diagnosed withmyeloproliferative neoplasms (MPNs)according to the WHO 2016 criteria in China, including 56 ET patients, 19 pre-PMF patients, and 43 overt PMF patients. Pre-PMF patients exhibited higher leukocyte counts [14.2(6.0-28.1) × 109/L vs 9.6(4.0-55.0) × 109/L, P = 0.003], LDH values [307(233-479)U/L vs 241(129-1182)U/L, P < 0.001], onset ages [67(32-76) years vs 50(16-79) years, P = 0.006], a higher frequency of splenomegaly(47.4% vs 16.7%, P = 0.018) and hypertension (57.9 vs 23.2%, P = 0.005) than ET patients. On the other hand, pre-PMF patients had higher platelet counts [960(500-2245) × 109/L vs 633(102-1720) × 109/L, P = 0.017], haemoglobin levels [152(115-174)g/L vs 119(71-200)g/L, P = 0.003], lower LDH values [307(233-479)U/L vs 439(134-8100)U/L, P = 0.007] and a lower frequency of splenomegaly(47.4 vs 75.6%, P = 0.031) than overt PMF patients. Next-generation sequencing landscape was performed in 50 patients, revealed the frequency of EP300 mutations was significantly increased in pre-PMF patients compared with ET and overt PMF patients (60 vs 10 vs 15.79%, P = 0.033), and WT1 was more often overexpressed (WT1/ABL1 copies ≥ 1.0%) in patients with overt PMF than in those with ET or pre-PMF(54.55 vs 16.67 vs 17.65%, P = 0.009). In terms of outcome, male sex, along with symptoms including MPN10, anaemia (haemoglobin < 120g/L), thrombocytopenia (platelet count < 100 × 109/L), leucocytosis (leukocyte counts > 13 × 109/L), high LDH value (> 350U/L), splenomegaly, WT1 overexpression(WT1/ABL1 copies ≥ 1.0%), KMT2A, ASXL1 and TP53 mutations, indicated a poor prognosis for PMF patients. The results of this study indicated that a comprehensive evaluation of BM features, clinical phenotypes, haematologic parameters, and molecular profiles is needed for the accurate diagnosis and treatment of ET, pre-PMF, and overt PMF patients.

Molecular Characterization of Clear Cell Renal Cell Carcinoma Reveals Prognostic Significance of Epithelial-mesenchymal Transition Gene Expression Signature

BackgroundThere is an ongoing need to develop prognostic biomarkers to improve the management of clear cell renal cell carcinoma (ccRCC). ObjectiveTo leverage enriched pathways in ccRCC to improve risk-stratification. Design, setting, and participantsWe retrospectively identified two complementary discovery cohorts of patients with ccRCC who underwent (1) radical nephrectomy (RNx) with inferior vena cava tumor thrombectomy (patients = 5, samples = 24) and (2) RNx for localized disease and developed recurrence versus no recurrence (n = 36). Patients with localized ccRCC (M0) in The Cancer Genome Atlas (TCGA, n = 386) were used for validation. Outcome measurements and statistical analysisA differential expression gene (DEG) analysis was performed on targeted RNA next-generation sequencing data from both discovery cohorts. Using TCGA for validation, Kaplan-Meier survival analysis and multivariable Cox proportional hazard testing were utilized to investigate the prognostic impact of DEGs, cell cycle proliferation (CCP), and a novel epithelial-mesenchymal transition (EMT) score on progression-free (PFS) and disease-specific (DSS) survival. Results and limitationsIn the discovery cohorts, we observed overexpression of WT1 and CCP genes in the tumor thrombus versus the primary tumor, as well as in patients with recurrence versus those without recurrence. A hallmark pathway analysis demonstrated enrichment of the EMT- and CCP-related pathways in patients with high WT1 expression in the TCGA (validation) ccRCC cohort. CCP and EMT scores were derived in the validation cohort, which was stratified into four risk groups using Youden Index cut points: CCPlow/EMTlow, CCPlow/EMThigh, CCPhigh/EMTlow, and CCPhigh/EMThigh. The CCPhigh/EMThigh risk group was associated with the worst PFS and DSS (both p < 0.001). In a multivariable analysis, CCPhigh/EMThigh was independently associated with poor PFS and DSS (hazard ratio = 4.6 and 10.3, respectively; p < 0.001). ConclusionsWe demonstrate the synergistic prognostic impact of EMT in tumors with a high CCP score. Our novel EMT score has the potential to improve risk stratification and provide potential novel therapeutic targets. Patient summaryGenes involved in epithelial-mesenchymal transition provides important prognostic information for patients with clear cell renal cell carcinoma.

A Moderate Increase in the WT1 mRNA Copy Number in Peripheral Blood of Patients with Long-Standing Acquired Aplastic Anemia without Apparent Signs of Myelodysplastic Syndrome

Background: The copy number of Wilms tumor 1 mRNA (WT1) correlates well with the acute myeloid leukemia (AML) cell burden and is being used as a reliable marker of residual AML cells. WT1 has also been shown to be useful for distinguishing high-risk myelodysplastic syndrome from low-risk myelodysplastic syndrome (MDS) as well as diagnosing disease progression with time in patients with low-risk MDS. However, few studies have so far focused on the role of measuring WT1 in diagnosing clonal evolution in patients with acquired aplastic anemia (AA). Although normal values of WT1 are defined as <50 copies/µgRNA, the reference values in AA patients without a propensity toward progression to MDS also remain unclear. To address these issues, we retrospectively studied 36 patients with bone marrow failure whose WT1 copy numbers in peripheral blood (PB) were serially measured to determine their correlation with the clinical picture and prognosis of the patients. Patients & Methods: PB samples of 36 patients (male/female, 19/17; a median age, 53.5 [range, 23-85] years) were subjected to WT1 measurement using a quantitative PCR method. The diagnoses of the patients were acquired AA in 29, MDS-multilineage dysplasia (MLD) evolved from AA in 4, and hemolytic paroxysmal nocturnal hemoglobinuria (PNH) in 3. Among 29 AA patients, 2 was untreated, and 22 were in convalescence while 5 were refractory after 0.5-457 months of immunosuppressive therapy (IST) and/or androgen therapy. WT1 was serially measured over time for all the patients except 5 whose initial examination produced negative (<50 copies/µgRNA) results. Myeloid malignancy-related gene abnormalities were screened in 11 patients showing an increase in the WT1 copy number. Results: WT1 at the initial examination was negative in 12 and positive in 17 (59%) patients with AA ranging from 59 to 520 copies. The 17 patients with increased WT1 had a disease duration of 122 months (11-495) but did not shown any signs of progression to MDS. The WT1 levels in patients possessing PNH-type cells (<50-330, median 62) were comparable to those without PNH-type cells (<67-520, median 89. There was a significant difference in the WT1 level between the 10 patients with a disease duration <3 years (<50-190, median <50) and the 19 patients with a disease duration ≥3 years (<50-520, median, 110, P=0.032). All 6 patients with WT1>200 had a disease duration ≥3 years. In contrast, the WT1 levels in the 4 patients who relapsed with cytopenia due to progression to MDS were as high as 690-5,700 (median, 2,400). Deep sequencing revealed somatic mutations of myeloid malignancy-related genes in 4 of 11 patients studied, which included EZH2 in an MDS patient (WT1 2,000), BCOR and DNMT3A in another MDS patient (WT1 2,800), and ASXL1 in a PNH patients (WT1 96). Conclusions: The WT1 copy numbers in PB of AA patients were higher than in AML patients with minimal residual diseases, and the cut-off value that helps diagnose MDS evolving from AA was considered to be around 600 copies. The high WT1 levels in long-standing AA patients without any signs of progression to MDS may represent an increased proliferative potential of hematopoietic stem/progenitor cells that support sustained hematopoiesis with their limited numbers. An increase in the WT1 copy number was associated with somatic mutations in some MDS patients but not in AA patients. Although AA patients with increased WT1 copy numbers need to be carefully followed, a moderate increase in the WT1 copy number does not portend myeloid malignancies unless the number steadily increases. DisclosuresIshiyama:Alexion Pharma GK: Research Funding.

WT1 Gene Expression in Peripheral Blood Before and After Allogeneic Stem Cell Transplantation is a Clinically Relevant Prognostic Marker in AML – A Single-center 14-year Experience

BackgroundThis work summarizes our experience with WT1 monitoring before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients and MethodsThe expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood according to the European Leukemia Net recommendations. Between May 2005 and August 2019, we analyzed 147 consecutive patients with acute myeloid leukemia with high WT1 expression at diagnosis, transplanted in first (CR1) or second (CR2) complete remission. ResultsAt the time of allo-HSCT, 107 patients had WT1-normal expression (WT1 ≤ 50 copies), and 40 patients had WT1-high expression. The median follow-up was 21 months. The estimated 5-year overall survival and event-free survival was significantly better in the WT1-normal cohort (65% and 57% vs. 37% and 25%; P = .0003 and P < .0001, respectively) and 5-year cumulative incidence of relapse was significantly lower in the WT1-normal group (19% vs. 53%; P < .0001). Five-year non-relapse mortality was not significantly different (20% and 23%). Multivariate analysis revealed WT1-high expression and acute graft-versus-host disease grade 3/4 as significantly negative prognostic factors for OS. Overall, 49 patients developed WT1 molecular relapse in the post-transplant period; in 14 cases, the therapeutic intervention was done. In all but 1 relapsed patient where WT1 minimal residual disease (MRD) was monitored (38 patients), we detected WT1-high levels (sensitivity of 97%). ConclusionThe results of the analysis confirmed our previous experience that WT1 status before allo-HSCT is a strong prognostic factor for both OS and relapse risk. In addition, we confirmed the usefulness of this marker for MRD monitoring after allo-HSCT. The main advantage is the possibility of frequent MRD monitoring in peripheral blood and early bone marrow examination based on WT1-high expression.

WT1 Expression in Peripheral Blood at Diagnosis and During the Course of Early Consolidation Treatment Correlates With Survival in Patients With Intermediate and Poor-Risk Acute Myeloid Leukemia

BackgroundUp to 55% of non-APL acute myeloid leukemias (AML) lack a molecular target suitable for standardized disease monitoring. We aimed to evaluate the prognostic significance of WT1 gene expression at early stages of intensive treatment. Patients and MethodsA total of 106 consecutive patients with intermediate and high-risk AML who had WT1 expression at diagnosis >500 copies/104ABL and who achieved remission after 1 to 2 cycles of induction treatment were analyzed. WT1 expression was measured in peripheral blood using a standardized European LeukemiaNet method. Overexpression was defined as >50 copies/104ABL. The median follow-up was 30 months. ResultsPatients with normal versus high WT1 expression after 2 cycles of chemotherapy had overall survival (OS) at 3 years of 66% versus 41% (P = .01); event-free survival (EFS) 45% versus 22% (P = .01). Prognostic significance of WT1 expression after 2 cycles of treatment was maintained in the group of patients treated with chemotherapy alone without hematopoietic stem cell transplantation in first line treatment (OS 70% vs. 36%, P = .02; EFS 35% vs. 0%, P = .03). Significant prognostic factors for EFS on multivariate analysis were the achievement of molecular remission (<50 copies of WT1) at any time during treatment (hazard ratio [HR] 0.47, P = .04) and increased WT1 expression after 2 cycles of chemotherapy (HR 2.0, P = .03). ConclusionIncreased WT1 expression after 2 cycles of chemotherapy is a negative prognostic factor for survival. WT1 remains a valuable molecular marker in AML without any leukemia-specific mutation, especially if next generation sequencing and/or digital polymerase chain reaction are not routinely available.

Prognostic Value of Ki67 Percentage, WT-1 Expression and p16/CDKN2A Deletion in Diffuse Malignant Peritoneal Mesothelioma: A Single-Centre Cohort Study.

Diffuse malignant peritoneal mesothelioma (DMPM) is a rare malignant neoplasm with a poor survival. Although some advances in knowledge have been obtained for the pleural form, much less is known about DMPM. Advantages in terms of prognosis are still limited and strong efforts need to be made. The aim of our study was to correlate several histological and molecular factors with survival in a large cohort of 45 DMPMs. We evaluated histotype, nuclear grade, mitotic count, necrosis, inflammation, desmoplastic reaction, Ki67 percentage, WT-1 expression, p16 protein by immunohistochemistry and CDKN2A deletion by FISH. Our results showed that epithelioid histotype, nuclear grade 2, mitotic count ≤5 x mm2, absence of desmoplasia and p16/CDKN2A deletion, low Ki67 value, and high WT-1 expression were correlated with the most prolonged survival (p = 0.0001). Moreover, p16 loss in immunohistochemistry reflected CDKN2A deletion detected with FISH, and both were correlated with the worst survival (p = 0.0001). At multivariate analysis, Ki67 value, WT-1 expression and p16/CDKN2A deletion emerged as independent prognostic factors (p = 0.01, p = 0.0001 and p = 0.01, respectively). These parameters are easy to analyse at the time of DMPM diagnosis and may support better patient stratification, prediction of treatment effectiveness and therapeutic optimization.

Subgroup Analysis Can Optimize the Relapse-Prediction Cutoff Value for WT1 Expression After Allogeneic Hematologic Stem Cell Transplantation in Acute Myeloid Leukemia

High WT1 expression after allogeneic hematologic stem cell transplantation (allo-HSCT) can strongly predict relapse in acute myeloid leukemia (AML). However, the cutoff values obtained have been inconsistent. Precise cutoff values may be optimized through subtype analysis; the RUNX1-RUNX1T1 fusion transcript provides an ideal reference. RUNX1-RUNX1T1 and WT1 transcript levels were simultaneously measured in 1299 bone marrow samples serially collected from 176 t(8;21) AML patients after allo-HSCT. The upper limit of the normal bone marrow WT1 level was 0.6%, which we previously reported to be the cutoff value for significant relapse prediction in AML as a whole. WT1 cutoff values of 0.6%, 1.2%, and 1.8% significantly differentiated patients in relapse after allo-HSCT. Nonetheless, patients with WT1 levels of 0.6% to 1.2% and those with levels of >1.2% and 1.8% after HSCT had rates of cumulative incidence of relapse similar to those with a continuous WT1 level of ≤0.6%, and both were significantly lower than that in patients with a WT1 level of >1.8%. WT1 expression was significantly related to RUNX1-RUNX1T1 transcript levels at WT1 levels of >1.8% but not at levels of 0.6% to 1.2% or >1.2% to 1.8%. Therefore, subgroup analysis can optimize the relapse-prediction cutoff value of WT1 expression. A cutoff level of 1.8% more accurately differentiates t(8;21) AML patients in relapse after allo-HSCT than does a cutoff level of 0.6%.

Significance of WT1 gene detection in the prognosis of acute myeloid leukemia patients with normal karyotype after hematopoietic stem cell transplantation

Objective To investigate the monitoring significance of WT1 gene level in the prognosis of acute myeloid leukemia (AML) patients with normal karyotype after hematopoietic stem cell transplantation (HSCT). Methods The clinical data of 115 AML patients with normal karyotype who were treated with HSCT from July 2009 to March 2017 in the First Affiliated Hospital of Soochow University were retrospectively analyzed. The dynamic detection of bone marrow WT1 gene was carried out by using reverse transcription- polymerase chain reaction (RT-PCR). According to the relative expression level median of WT1 gene before transplantation, the whole patients were divided into the two groups (< median group and ≥ median group) for survival analysis. Results There were 52 males and 63 females in 115 patients. The average age was (39±10) years old. The median white blood cell count at initial diagnosis was 20.45×109/L [(0.5-355.9)×109/L], the ratio of blast cells in the bone marrow was 0.60±0.28, and the relative expression level median of WT1 gene was 87×104, while the median time of the follow-up was 24 (3-79) months. Among 115 patients, 19 cases relapsed. Remission group (96 cases) and relapse group (19 cases) were followed up. The WT1 gene level was monitored by using bone marrow puncture in 1 month, 3 months, 6 months, 12 months after transplantation. It was found that the WT1 gene relative expression level of relapse group was higher than that of remission group, and the differences between the two groups at 6 month-point [remission group (187±50)×104, relapse group (871±211)×104, t= 2.519, P= 0.014] and 12 month-point [remission group (51±9)×104, relapse group (1 797±312)×104, t= 4.000, P < 0.05] were statistically different. The overall survival (OS) rate of 2-year, progression-free survival rate in WT1 gene relative expression level < 87×104 group were higher than those in WT1 gene relative expression level ≥ 87×104 group, the relapse rate in WT1 gene relative expression level < 87×104 group was lower than that in WT1 gene relative expression level ≥ 87×104 group, and the differences were statistically different (all P < 0.05). Multivariate analysis showed that the level of WT1 gene at 12 months after transplantation was an independent factor affecting OS (HR = 4.12, P = 0.046) and PFS (HR = 5.95, P = 0.001). There were 19 cases of recurrence (16.5%), with a median relapsed time of 11 (1-60) months. When WT1 gene relative expression level was significantly increased in 19 patients, firstly immunosuppressive agents were reduced, of which 6 patients were not influenced by this intervention; in other 13 cases, only 5 cases were influenced by intervention. Conclusions For CN-AML patients, the expression level of WT1 gene before transplantation has a negative correlation with the prognosis. The expression level of WT1 gene at 12 months after transplantation is an independent factor for affecting the survival. The relapsed patients have a higher WT1 expression level, and clinical interventions for relapsed patients have a favorable effect. Key words: Gene, WT1; Hematopoietic stem cell transplantation; Leukemia, myeloid, acute; Prognosis

Quantitative proteomics identifies brain acid soluble protein 1 (BASP1) as a prognostic biomarker candidate in pancreatic cancer tissue.

BackgroundPancreatic cancer is a heterogenous disease with a poor prognosis. This study aimed to discover and validate prognostic tissue biomarkers in pancreatic cancer using a mass spectrometry (MS) based proteomics approach.MethodsGlobal protein sequencing of fresh frozen pancreatic cancer and healthy pancreas tissue samples was conducted by MS to discover potential protein biomarkers. Selected candidate proteins were further verified by targeted proteomics using parallel reaction monitoring (PRM). The expression of biomarker candidates was validated by immunohistochemistry in a large tissue microarray (TMA) cohort of 141 patients with resectable pancreatic cancer. Kaplan-Meier and Cox proportional hazard modelling was used to investigate the prognostic utility of candidate protein markers.FindingsIn the initial MS-discovery phase, 165 proteins were identified as potential biomarkers. In the subsequent MS-verification phase, a panel of 45 candidate proteins was verified by the development of a PRM assay. Brain acid soluble protein 1 (BASP1) was identified as a new biomarker candidate for pancreatic cancer possessing largely unknown biological and clinical functions and was selected for further analysis. Importantly, bioinformatic analysis indicated that BASP1 interacts with Wilms tumour protein (WT1) in pancreatic cancer. TMA-based immunohistochemistry analysis showed that BASP1 was an independent predictor of prolonged survival (HR 0.468, 95% CI 0.257–0.852, p = .013) and predicted favourable response to adjuvant chemotherapy, whereas WT1 indicated a worsened survival (HR 1.636, 95% CI 1.083–2.473, p = .019) and resistance to chemotherapy. Interaction analysis showed that patients with negative BASP1 and high WT1 expression had the poorest outcome (HR 3.536, 95% CI 1.336–9.362, p = .011).InterpretationWe here describe an MS-based proteomics platform for developing biomarkers for pancreatic cancer. Bioinformatic analysis and clinical data from our study suggest that BASP1 and its putative interaction partner WT1 can be used as biomarkers for predicting outcomes in pancreatic cancer patients.

WT1 gene is overexpressed in myeloproliferative neoplasms, especially in myelofibrosis

Classical Philadelphia-negative myeloproliferative neoplasms include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). They are characterized by the presence of driver mutations of JAK2, CALR or MPL genes. Overexpression of WT1 is used as a marker of minimal residual disease in acute myeloid leukemia, especially after allogeneic stem cell transplantation (SCT). We investigated WT1 expression at diagnosis in 152 MPN patients and showed that the WT1 transcript was overexpressed in PMFs and PVs compared to controls. In particular, WT1 transcript levels were higher in PMF than in ET and PV. WT1 transcript levels were significantly increased during myelofibrotic transformation of ET or PV. Using multivariate linear regression, high WT1 transcript levels in PMF were associated with age over 65, splenomegaly and thrombocytopenia. The ROC curve analysis showed that a level of WT1 transcript >10 WT1 copies/104ABL1 enabled the diagnosis of PMF with a specificity of 95.8% (PMF vs ET; ROC AUC = 0.91). In myelofibrosis, studying follow-ups of WT1 transcript showed that this marker is of interest after allogeneic SCT. These results demonstrate that WT1 overexpression is a simple marker of myelofibrosis in MPN and could be used during patient follow-up.