WT1 Gene Research Articles

Analysis of age-specific cytogenetic changes among 515 patients withacute myeloid leukemia

To characterize cytogenetic changes and prognosis of patients with acute myeloid leukemia (AML) from different age groups. The karyotypes of 515 AML patients were analyzed by using short-term culture of bone marrow cells and R-banding technique. Combined with FAB typing and genetic testing, cytogenetic changes and prognosis of different age groups were analyzed. The abnormal cloning rate was 54.6% among the 515 patients. The abnormal cloning rate and adverse risk karyotype proportion of those with myeloproliferative syndromes (MDS) and secondary AML were higher than those with de novo AML (P = 0.027; P<0.01). A significant difference was found in the number of structural abnormalities and proportion of favorable risk karyotypes among different age groups (P = 0.026; P = 0.004). And there was also a significant difference in the abnormal cloning rate between different FAB types (P<0.01). In those with non-acute promyelocytic leukemia (APL), the expression level of WT1 gene seemed to affect the prognosis. The survival rate of patients with karyotypes of adverse risk was lower than those with karyotypes of favorable risk (P = 0.015). The survival rate of the ≥60-year-old group was lower than the ≤30-year-old and 31 to 59-year-old groups (P<0.01, P<0.01). The karyotypes of AML patients have different age distribution characteristics. The survival rate of ≥60-years-old group and karyotype of poor prognosis is low. Patients with MDS with secondary AML have a poor prognosis.

Thymoquinone induces cell proliferation inhibition and apoptosis in acute myeloid leukemia cells: role of apoptosis-related WT1 and BCL2 genes

Acute myeloid leukemia (AML) is an aggressive and heterogeneous disease characterized by an abnormal proliferation and impaired differentiation of the myeloid precursor cells. The outcome for most AML patients remains poor with high relapse rates and chemotherapy remains the first line treatment for AML. The Wilms tumor wt1 and the anti-apoptotic BCL2 genes are upregulated in AML and are known to be involved in apoptosis inhibition. In the present study we evaluated the molecular mechanisms underlie the anti-proliferative and pro-apoptotic activities exerted by thymoquinone (TQ), the major biologically active compound of the black seed oil on acute myeloid leukemia (AML) cell line-HL60. Cell proliferation was determined by WST-1 assay and apoptosis rate was assessed by flow cytometry using annexin-V/7AAD staining. The expression of target genes was analyzed by real-time RT–PCR analysis. TQ significantly reduced HL60 cell viability and induced apoptosis in a dose and time-dependent manner. In order to decipher the molecular mechanisms underlie the anti-cancer activities induced by TQ in AML cells, we investigated its effect on the expression of WT1 and BCL2 genes. TQ significantly decreased the expression of WT1 and BCL2 genes in a dose and time-dependent manner. In summary, these findings suggest that TQ induces cell proliferation inhibition and apoptosis in acute myeloid leukemia cells most likely through targeting the apoptosis-related WT1 and BCL2 genes and also suggest that TQ could be a promising strategy for AML therapy.

PB1711 MUTATIONAL ANALYSIS WILMS TUMOR 1 GENE IN DE NOVO CASES OF ACUTE MYELOID LEUKAEMIA; PREVALENCE, CORRELATION AND REPORT OF NOVEL MUTATION IN INDIAN POPULATION

Background:Cytogenetic and molecular abnormalities associated with pathogenesis of acute myeloid leukemia (AML) are broad and heterogeneous and acts as a cardinal marker for AML classification, prognostication and risk stratification. Mutation of the Wilms tumour 1 gene (WT1) is relatively frequent in leukemia and has been found to be present in about 10% &gt; 15% of cases of AML mainly affecting exons 7 and 9, and less frequently exons 1, 2, 3, and 8. Different studies show variable and conflicting results with few showing poor prognosis in cases of AML due to WT1 overexpression or mutation, while others suggesting that WT1 has no significant impact on the prognosis of AML.Aims:The objective of this pilot study was to evaluate the prevalence of WT1 gene mutation in de novo cases of AML and its correlation with clinical features and prognosis.Methods:Study was conducted at All India Institute of Medical Sciences, New Delhi in 100 de novo cases of AML across the all age groups. Bone marrow aspirates (BMA) or peripheral blood (PB) sample was collected from diagnosed cases of AML and were processed for genomic DNA extraction. Genomic DNA amplified by polymerase chain reaction (PCR) for targeted regions exon 7 and 9 of WT1 gene using designed primer pair (forward and reverse for both exon 7 and 9). The PCR amplified product was then subjected to direct Sanger sequencing using genetic analyzer 3100/3700 (Applied bio‐system, USA). The targeted gene sequence results were then analyzed using Chromas© 2.6 software tool. The statistical analysis of data was done using Pearson's chi‐squared test (χ2) and Fisher's exact test.Results:1. The WT1 point mutation (substitution) was identified in two cases (2%) aged 15 and 28 years respectively on exon 9 of WT1 gene. One of these two mutations was novel and has not been previously reported.2. Twelve cases (12%) were found to have synonymous single nucleotide polymorphism (SNP) in exon 7 which has been previously reported in SNP database (rs16754) as heterozygous in nature (WT1 A→G). 3. Blast percentage was &gt;50% in both the cases harbouring WT1 mutation (mutant cases). However, there were no statistically significant differences in other clinical and haematological parameters like haemoglobin, white blood cells (WBCs) count and platelets counts between wild type and mutant cases.4. Both mutant cases were young adults, relapsed after consolidation and eventually met early death compared to wild type cases.Summary/Conclusion:Only few case studies on WT1 gene mutation in AML have been reported and there is lack of such study and precise published data from India to date. From this pilot study we conclude that WT1 mutation is not so prevalent among de novo cases of AML in Indian population. Correlation between WT1 mutation and clinical outcome of the cases suggested that mutant cases had poor clinical outcome and early death. However, this study was limited by a relatively small sample size and incorporating only the mutational hotspot region of the gene. In future we need to perform this study on larger sample size together with study of whole WT1 gene by next generation sequencing (NGS) in order to reach any statistically significant conclusion.

PF265 GENE MUTATION PROFILE IN PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA (APL)

Background:Though survival rates have improved in majority of APL patients, some high‐risk or intermediate risk patients still encounter early mortality. The underlying genetic mechanism between the different risk groups remains incompletely understood, therefore bringing necessity to explore.Aims:The aim of this study was to identify mutations in a panel of 114 leukemia‐associated genes in newly diagnosed APL patients and to analyze the correlation between gene mutations and APL patients of different risk groups.Methods:Forty‐six newly diagnosed APL patients were enrolled between October 2015 and December 2018. Mutational assessment of 114 genes was performed using next‐generation sequencing approach. Induction treatment started with all‐trans retinoic acid (ATRA) and arsenic trioxide based double induction. Anthracycline was added when peripheral white blood cell (WBC) ≥10X109/L. Statistical comparisons were made between gene mutations and APL patients of different risk groups (high risk‐ HR, intermediate risk‐ IR and low risk‐ LR).Correlations between gene mutations and patients suffered from early death (ED, defined as patients who died during induction treatment) and patients reached complete remission (CR) were further investigated.Results:Of the 46 APL patients, there were 26.09% (12/46), 54.35% (25/46) and 19.57% (9/46) were HR, IR and LR patients, respectively. Six patients (13.04%, including 3 HR and 3 IR patients) died during induction treatment, while all of the other 40 patients achieved CR by the end of induction treatment. One or more mutations were detected in 91.30% of the patients (42/46). A total of 102 mutations in 52 genes were detected with the median number of gene mutations was 2 (range 0–6) and mutation rate ≥10%. Though it seemed that HR patients had the highest frequency of mutations (median number 3, range 1–5), LR patients had the lowest frequency of mutations (median number 1, range 0–4), and the median mutation number in IR patients was 2 (range 0–6), there were no significant difference between the three risk groups regarding the median gene mutation numbers (P&gt;0.05).There were 5 frequent mutations identified, including FLT3 (36.96%, 17/46), WT1 (15.22%, 7/46), FAT1 (13.04%, 6/46), ARIDIA (13.04%, 6/46), SETBP1 (8.70%, 4/46), etc. A total of 13 mutations in 12 genes including FLT3, WT1, NRAS, KRAS and NOTCH1 etc were detected in 6 ED patients. The median number of gene mutations in ED patients was 2 (range 1–4), which did not show difference with that in non‐ED patients (median number 2, range 0–6). Among these mutations, only NRAS gene mutations were detected in two ED patients (1 HR and 1 IR patients), which was the same mutation NRAS G12C. FLT3 gene mutations (including FLT3‐ITD and FLT3‐TKD) were the most frequently found in HR APL patients. 88.33% of HR patients(10/12) harbored FLT3 mutations, while 20% of IR patients (5/25) and 22.22% of LR patients (2/9) were found harbored FLT3 mutations(P &lt; 0.05). Among the 17 cases of FLT3 mutant patients, only one HR patient (5.88%, 1/17), who carried FLT3‐TKD D835Y mutation, died 3 days after induction treatment. Meanwhile, the less common WT1 gene mutations were all found in IR APL patients.Summary/Conclusion:HR APL patients had the highest frequency of gene mutations, however there were no significant difference between the three risk groups regarding the median mutation numbers. ED patients did not have more gene mutations compared with non‐ED patients. FLT3 gene mutations were the most frequently occurred mutations, especially in HR APL patients, though might have no relation with early mortality.

PB1726 DOUBLE FLUDARABINE‐BASED INDUCTION AND INFECTIVE RISK: THE BOLOGNA EXPERIENCE.

Background:“3+7” represents the current standard of care for acute myeloid leukemia (AML). Fludarabine‐based regimens have limited application due to the absence of survival benefit and prolonged hematological toxicity after 2nd cycle. Infections in neutropenic patients represent an important cause of mortality and morbidity.Aims:To analyze the infective risk associated with intensive chemotherapy based on double‐FLAI induction.Methods:We retrospectively collected data of 40 consecutive adult patients with newly diagnosed AML treated with FLAI‐5 induction therapy (Fludarabine IV 25 mg/sqm/die days 1‐5, Cytarabine IV 2000 mg/sqm/die days 1‐5 and Idarubicin 10 mg/sqm/die on days 1, 3 and 5). Patients in complete remission (CR) proceeded to second FLAI‐5 followed by allogeneic transplant or chemotherapy consolidation. The incidence and type of infective events were collected in 36/40 patients. Patients were compared with a historical court of 39 patients treated with non‐fludarabine based induction. MRD was assessed by qRT‐PCR or WT1 gene expression. Data were harmonized using RedCap custom eCRFs.Results:Forty patients received FLAI‐5 from August 2015 to December 2018. Median age at diagnosis was 53 years (range 19‐66). According to NCCN‐2016 stratification criteria 12/40 (30%) patients were favorable, 10/40 (25%) intermediate and 17/40 (42.5%) high risk; one patient wasn’t evaluable. Thirty‐five patients (87.5%) achieved a CR after induction, 4 (10%) patients were refractory and 1 patient (2.5%) died during induction. Twenty‐nine of 40 patients were evaluable for MRD analysis and 15/29 (52%) patients achieved MRD negativity. Twenty‐two of the 35 patients in CR proceeded to 2nd FLAI‐5 chemotherapy regimen.At induction, patients recovered from neutropenia (ANC&gt; 500 x 109/L) after 21.5 days (range 13‐32) from the end of chemotherapy and reached platelet &gt; 20 x 109/L after a median of 16 days (range 10‐33). Patients spent a median of 33 nights in hospital (range 14‐61; historical control 35 nights, range 31‐42; p = ns). After 2nd cycle, median days for ANC and platelet recovery were 29 and 26 (range 16‐45 and 13‐60 respectively, historical control for ANC recovery 20 days, range 18‐25; p = 0.001). Median patients’ hospitalization was 39 nights (range 26‐55; historical control 32.5 nights, range 24‐47; p = 0.003).Patients and historical controls received same antimicrobial prophylaxis in induction and experienced respectively 33/36 (91.7%) and 36/39 (92.3%) infective events. Patients had a higher number of infections of unknown origin (15/33 vs 6/36, p. 002), and lower incidence of Gram‐positive isolates (4/12 vs 15/22, p = 0.002). Any‐mould infection rate was comparable in the two groups.Twenty‐seven of 36 patients and 22/39 controls proceeded to consolidation chemotherapy. Antimould prophylaxis was different, with patients receiving mainly posaconazole 17/27 (63 %), fluconazole 5/27 (18.5%), itraconazole 4/27 (14.8%) and voriconazole 1/27 (3.7%); while controls received posaconazole 4/22 (18.2%) fluconazole 5/22 (22.7%), itraconazole 12/22 (54.5%) and voriconazole 1/22 (4.5%). Twenty‐two of 27 patients (81.5%) and 17/22 (77.3%) in controls had an infection. Three patients of 27 had a probable mould infection and 1 possible, compared to 1 proven mould infection and 1 possible in the 22 controls (any‐mould infection 4/27 vs 2/22).Summary/Conclusion:In our experience FLAI‐5 is associated with high CR rates, but prolonged neutropenia and hospitalization after 1st consolidation. However, this doesn’t translate in a higher infection risk in this set of patients.

An integrative approach reveals genetic complexity and epigenetic perturbation in acute promyelocytic leukemia: a single institution experience

Acute promyelocytic leukemia (APL) is a distinct type of acute myeloid leukemia that is defined by the presence of the translocations that mostly involve the RARA gene. The most frequent translocation is the t(15;17), which fuses the RARA gene with the PML gene. Previous studies have shown that other cooperative mutations are required for the development of APL after the initiating event of the t(15;17). In this study, we combined cytogenetics with next-generation sequencing and single-nucleotide polymorphism array to study the genetic complexity in 20 APL cases diagnosed in our institution. All but 3 cases had additional genetic aberrations. Our study demonstrated that somatic mutations are frequent events in APL. In addition to the previously reported recurrent cooperative mutations in the FLT3, WT1, and RAS genes, we identified mutations in several epigenetic modifiers, including TET2, EZH2, and DNMT3A, co-occurring with either FLT3 or WT1 mutations. Mutations of the WT1 gene and chromosome 11p copy neutral loss of heterozygosity affecting WT1 are present in a third of the cases in our series. Two-thirds of APL cases in our study demonstrated a global reduction but focal accumulation of H3K27 methylase (H3K27me) expression, indicating a disorganized chromatin methylation pattern with generally more accessible chromatin status. Our study confirmed genetic complexity of APL and revealed that epigenetic aberrations are more common than previously expected. Although epigenetic modulation is not a common treatment strategy in APL, targeting this pathway may have some clinical utility in refractory or relapsed APL cases.

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

SAT-302 A Rare Diagnosis in a Female Adolescent Presenting with Primary Amenorrhea

Background Primary amenorrhea (PA) occurs in about 2% of women in the reproductive age group. Evaluation of PA can be challenging, due to the multiple possible etiologies. In patients with PA secondary to hypergonadotropic hypogonadism, chromosomal anomalies are common. Clinical Case A 16 9/12 years old female with early kidney failure and delayed puberty with primary amenorrhea, in whom phenotypic sex and gender identity were not consistent with genotypic sex. Physical exam revealed a normal phenotypic female with no dysmorphic features. She has Tanner II-III for breast development, normal female external genitalia, Tanner V for pubic hair, no clitoromegaly and no galactorrhea. Workup demonstrated hypergonadotropic hypogonadism with a 46,XY karyotype. The SRY gene was present on FISH analysis. Patient had low levels of estradiol, testosterone, AMH and Inhibin, and normal prolactin, thyroid and adrenal function. MRI revealed duplicated internal female genital organs and unilateral streak gonad. DEXA scan showed severe osteopenia. The gender identity was female and consistent with the sex of rearing. The patient was started on weekly 0.050 mg Estradiol transdermal patch with gradual titration and will be followed with cyclical estrogen-progesterone replacement therapy. Bilateral gonadectomy was done. Surgical pathology report consistent with bilateral streak gonads. Conclusion Clinical and biological evaluation corroborated with the significant past medical history led to the highly suspected diagnosis of Denys-Drash Syndrome (DDS), a very rare genetic syndrome with approximately 150 cases reported worldwide. It is important to consider DDS and test for WT1 gene mutations in any patient with early onset kidney failure caused by diffuse mesangial sclerosis. Karyotype should be obtained in all patients at diagnosis of DDS, even in the absence of ambiguous genitalia. In 46, XY patients, performing gonadectomy at the time of nephrectomy is best, in order to prevent malignancy of streak gonads. Early institution of hormonal therapy is essential to induce puberty and prevent osteoporosis. If not evaluated for early, patients who present with normal female phenotypes, despite non-functional gonads, will not present until adolescence with a chief complaint of primary amenorrhea. Reference: Day YL, Fu JF et al. WT1 mutation as a cause of 46 XY DSD and Wilm's tumor: a case report and literature review. Acta Pediatr 2011. Jul: 100(7): e 39-42. Ostrer H. Disorders of sex development (DSDs): an update. J Clin Endocrinol Metab. 2014 May;99(5): 1503-9

Desmoplastic Small Round Cell Tumor

Desmoplastic small round cell tumor (DSRCT) is a relatively recently identified, rare, aggressive cancer that arises from the peritoneal lining of the abdominal cavity and predominantly affects male adolescents and young adults. DSRCT is included in the Ewing family of tumors and harbors a unique translocation between EWSR and WT1 genes. Patients characteristically present with advanced disease, including widespread involvement of the abdominal cavity and pelvis, with frequent extraperitoneal metastases involving liver, spleen, and thoracic lymph nodes. Five-year overall survival remains approximately 15 to 20% in recent series despite aggressive multimodal therapy. Neoadjuvant chemotherapy, complete resection, defined as less than 1 cm3 of residual tumor, and consolidative whole abdominopelvic radiotherapy (WAP-RT) appear to play important role in curative treatment. The rarity of DSRCT makes large randomized trials difficult and consensus statements about clinical approach impossible. However, this chapter summarizes the best available data on the pathogenesis, diagnosis, and treatment of DSRCT; highlights the major advances made in these areas; and discusses the addition of local therapy to the treatment paradigm. This review contains 3 figures, 1 table, and 51 references. Key Words: DSRCT, peritoneal malignancy, desmoplastic round cell tumor, surface malignancies of the peritoneum, desmoplastic small round cell tumor of the abdomen, surgical oncology, rare malignancies, surgical therapy of abdominal malignancies

Desmoplastic Small Round Cell Tumor

Desmoplastic small round cell tumor (DSRCT) is a relatively recently identified, rare, aggressive cancer that arises from the peritoneal lining of the abdominal cavity and predominantly affects male adolescents and young adults. DSRCT is included in the Ewing family of tumors and harbors a unique translocation between EWSR and WT1 genes. Patients characteristically present with advanced disease, including widespread involvement of the abdominal cavity and pelvis, with frequent extraperitoneal metastases involving liver, spleen, and thoracic lymph nodes. Five-year overall survival remains approximately 15 to 20% in recent series despite aggressive multimodal therapy. Neoadjuvant chemotherapy, complete resection, defined as less than 1 cm3 of residual tumor, and consolidative whole abdominopelvic radiotherapy (WAP-RT) appear to play important role in curative treatment. The rarity of DSRCT makes large randomized trials difficult and consensus statements about clinical approach impossible. However, this chapter summarizes the best available data on the pathogenesis, diagnosis, and treatment of DSRCT; highlights the major advances made in these areas; and discusses the addition of local therapy to the treatment paradigm. This review contains 3 figures, 1 table, and 51 references. Key Words: DSRCT, peritoneal malignancy, desmoplastic round cell tumor, surface malignancies of the peritoneum, desmoplastic small round cell tumor of the abdomen, surgical oncology, rare malignancies, surgical therapy of abdominal malignancies

Long-term outcome in a case series of Denys-Drash syndrome.

BackgroundDenys–Drash syndrome (DDS) is a rare disease caused by mutations in exons 8 and 9 of the WT1 gene. It is characterized by the association of early onset steroid-resistant nephrotic syndrome (SRNS), Wilms’ tumour and, in some patients, intersex disorders, with increasing risk of gonadoblastoma. There are few published data concerning the long-term outcome of patients with DDS. The aim of this study was to report our experience. MethodsData were collected from five children (three boys) with confirmed DDS diagnosed from 1996 to 2017. The mean follow-up of these patients was 16 years.ResultsThe patients presented with SRNS and diffuse mesangial sclerosis at renal biopsy. All patients were hypertensive and progressed to end-stage kidney disease, initiating dialysis at a mean age of 28 months. Three patients developed Wilms’ tumour 9 months after the SRNS was identified, which was treated by nephrectomy and chemotherapy. All five patients received kidney transplantation. SRNS did not recur after transplantation in any of the patients and graft survival was similar to that of other kidney transplant recipients in our programme. All three boys had ambiguous genitalia and cryptorchidism but a confirmed male karyotype (46, XY). One girl presented with gonadal agenesis, whereas the other one had normal female ovarian tissue and external genitalia. Both girls had a female karyotype (46, XX). Gonadoblastoma was not observed at any case.ConclusionsEarly DDS recognition in patients with SRNS is crucial due to its low prevalence, the specific treatment approach required and early detection of Wilms’ tumour. Few data are available regarding long-term outcomes.

Correlations between Histological and Array Comparative Genomic Hybridization Characterizations of Wilms Tumor.

Wilms tumor, or nephroblastoma, is the most common pediatric renal malignancy. Its diagnosis is principally based on histology. Several genetic loci have been shown to be associated with Wilms tumor formation, including WT1, WT2, FWT1, FWT2, CTNNB1, WTX, and TP53. Other loci, such as 1p, 2q, 7p, 9q, 12q, 14q, 16q, 17p, and 22, have also been implicated in the etiology of Wilms tumor. The aim of this study is to elucidate the molecular pathogenesis of this tumor. In the present study, we analyzed the histological appearance and copy number aberrations using array comparative genomic hybridization of six Wilms tumors without somatic mutation in the WT1 gene. Many chromosomal aberrations on array comparative genomic hybridization analysis revealed that the genetics of Wilms tumors are extremely complex. Amplifications and deletions of large DNA fragments were observed in some samples. Amplifications of NDUFV1, ZIC2, SIX1, NR2F2, MIR1469, SOX9, JAG1, MIR6870, and GNAS were found in all six Wilms tumors. Moreover, amplifications of five genes were identified in the Wilms tumors of stromal type and amplifications of at least 10 genes were identified in the Wilms tumors of epithelial type. Our results indicated that amplifications of nine genes are the essential events in the tumorigenesis of Wilms tumor, which may inform its clinical and therapeutic management. In addition, mixed type Wilms tumor may be the heterogeneous group able to be classified using genetic results of epithelial and stromal components based on immunohistochemistry.

WT1 Gene Overexpression in Differential Diagnosis of Ph-Negative Myeloproliferative Disorders

Aim. To assess the rate of WT1 gene overexpression and its clinical value in Ph-negative myeloproliferative disorders (MPD). Materials &amp; Methods. The trial included 72 patents with Ph-negative MPD. Among them there were patients with primary myelofibrosis (MF; n = 32), post-polycythemia vera MF (n = 7), polycythemia vera (PV; n = 17), and essential thrombocythemia (ET; n = 16) with median age of 57 years (range 19-78 years). Median (range) time from diagnosis to the date of evaluating WT1 expression in PV, ET, and MF was 9.4 (0-309), 14.4 (0-55), and 21.4 months (0-271 months), respectively. WT1 expression in terms of WT1 copies/10&lt;sup&gt;4&lt;/sup&gt; ABL copies was measured by quantitative PCR. Results. WT1 gene overexpression is revealed solely in patients with MF (in 34/39; 87 %). In PV/ET no WT1 gene overexpression was observed. Median WT1 expression in MF was 230/10&lt;sup&gt;4&lt;/sup&gt; ABL copies (range 42.2-9,316.45/10&lt;sup&gt;4&lt;/sup&gt; ABL copies). Sensitivity and specificity of WT1 gene overexpression in MF with respect to PV/ET were 87 % and 100 %, respectively. A distinct correlation was identified between WT1 gene expression level and spleen size, duration of the disease, blast cell count, and DIPSS risk group. WT1 gene expression level could be correlated neither with age and sex, nor with MF mutation status and leucocyte, thrombocyte, and haemoglobin levels. Conclusion It appears that due to a high specificity and sensitivity of WT1 gene expression in MF it can be used as a marker for differential diagnosis of Ph-negative MPD. A correlation between WT1 gene expression and tumor mass in MF cannot be excluded. It is advisable to analyze the dynamics of WT1 expression level to predict the efficacy of current targeted therapy.

Epigenetic deregulations of Wnt/β-catenin and transforming growth factor beta-Smad pathways in esophageal cancer: Outcome of DNA methylation.

Promoter methylation of tumor suppressor genes (TSGs) is a well-reported portent in carcinogenesis; hence, it is worthy to investigate this in high-risk Northeast population of India. The study was designed to investigate methylation status of 94 TSGs in esophageal squamous cell carcinoma (ESCC). Further, the effect of OPCML promoter methylation on gene expression was analyzed by immunohistochemistry. Moreover, in silico protein-protein interactions were examined among 8 TSGs identified in the present study and 23 epigenetically regulated genes reported previously by our group in ESCC. Methylation profiling was carried out by polymerase chain reaction array and OPCML protein expression was examined by tissue microarray-based immunohistochemistry. OPCML, NEUROG1, TERT, and WT1 genes were found hypermethylated and SCGB3A1, CDH1, THBS1, and VEGFA were hypomethylated in Grade 2 tumor. No significant change in OPCML expression was observed among control, Grade 1, and Grade 2 tumor. Conclusively, hypermethylation of the studied OPCML promoter in Grade 2 tumor produced no effect on expression. Unexpectedly, OPCML expression was downregulated in Grade 3 tumor in comparison to other groups signifying that downregulation of OPCML expression may lead to higher grade of tumor formation at the time of diagnosis of ESCC in patients. Significant interactions at protein level were found as VEGFA:PTK2, CTNNB1:CDH1, CTNNB1:VEGFA, CTNNB1:NEUROG1, CTNND2:CDH1, and CTNNB1:TERT. These interactions are pertinent to Wnt/β-catenin and TGF-β-Smad pathways. Deranged OPCML expression may lead to high-grade ESCC as well as epigenetically regulated genes, that is, CDH1, CTNNB1, CTNND2, THBS1, PTK2, WT1, OPCML, TGFB1, and SMAD4 may alter the Wnt/β-catenin and TGF-β-Smad pathways in ESCC. Further study of these genes could be useful to understand the molecular pathology of ESCC with respect to epithelial-mesenchymal transition (EMT) mediated by Wnt/β-catenin and TGF-β signaling pathways.

Prognostic Impact of WT-1 and Survivin Gene Expression in Acute Myeloid Leukemia Patients.

Wilms Tumor 1 (WT1) and Survivin genes are important leukemia-associated antigens (LAAs) in AML with potential prognostic impact. We investigated WT1 and Survivin expression levels by RT-PCR in 61 AML patients in correlation with clinical characteristics and outcomes. WT1 was overexpressed in 45 patients (73.8%), associated with higher BM blasts (p = 0.017), lower incidence of favorable-prognosis cytogenetics (p = 0.035), and higher incidence of Flt3-ITD mutations (p = 0.026). Survivin was overexpressed in 17 patients (27.9%) with higher mean WBC count (p = 0.049). Patients with overexpression of either gene showed inferior complete remission (CR) rates and survival rates, patients with overexpression of both genes showed higher mean WBCs (p = 0.035) and higher BM blasts (p = 0.029) while the double negative group showed higher incidence of favorable cytogenetic events (p = 0.021), better CR rates and survival rates. Our findings support the introduced prognostic impact of WT1 and Survivin genes in AML patients and its potential use in MRD monitoring and immunotherapy.

Prediction of FLAG ± Ida Regimen Efficacy in Patients with Relapsed/ Refractory Acute Myeloid Leukemia

Aim. To assess the efficacy of FLAG/FLAG-Ida regimen and to identify factors that influence remission, duration of disease-free survival (DFS) and overall survival (OS) of patients with relapsed/refractory acute myeloid leukemia (AML). Materials &amp; Methods. The trial included 54 patients (28 men and 26 women), median age was 37 years (range 18-70 years). 27 (50 %) out of 54 patients had refractory AML and 27 (50 %) patients had relapsed AML. FLAG and FLAG-Ida regimens were administered as induction therapy. 37 (68.5 %) patients received bone marrow transplantation. Molecular genetic and cytogenetic examinations were performed prior to therapy and on the 28&lt;sup&gt;th&lt;/sup&gt; day after the start of treatment. WT1 gene expression was evaluated on the 14&lt;sup&gt;th&lt;/sup&gt;-16&lt;sup&gt;th&lt;/sup&gt; day of treatment. Results. Complete remission (CR) was achieved in 42 (77.8 %) out of 54 patients. Refractoriness to therapy was observed in 9 (16.7 %) out of 54 patients, mortality was 5.5 % (3/54). Remission rate was higher in patients with relapsed AML compared with refractory AML: 85.2 % (23/27) and 70.4 % (19/27), respectively. On the 14&lt;sup&gt;th&lt;/sup&gt;-16&lt;sup&gt;th&lt;/sup&gt; day of treatment patients with blast cell count ≥ 10 % in bone marrow (BM) showed significantly lower CR rate (60 %) compared with the group of patients with &lt; 10 % blast cells in BM (89.6 %; p = 0.024) and shorter DFS (median 7.6 vs. 17.6 months, respectively; p = 0.03). Median DFS in patients with WT1 expression reduction to &lt; 1 log on the 14&lt;sup&gt;th&lt;/sup&gt;-16&lt;sup&gt;th&lt;/sup&gt; day was 5 vs. 18 months in patients without WT1 expression reduction (p = 0.01). DFS varied in groups of patients with blast cell count &lt; 10 % in BM on the 14&lt;sup&gt;th&lt;/sup&gt;-16&lt;sup&gt;th&lt;/sup&gt; day of treatment based on the level of WT1 expression reduction (p = 0.04). MRD-negative patients (57.1 %) showed significantly longer DFS and OS compared with MRD-positive patients (median DFS was 17.6 vs. 5.2 months, respectively, p = 0.02; median OS was 19 vs. 6.9 months, p = 0.0002). Median DFS and OS were different only in ELN low- and high-risk groups (median not reached vs. 5.2 months, respectively, p = 0.039; median not reached vs. 10.2 months, p = 0.039). Conclusion. FLAG and FLAG-Ida are effective and safe regimens in the treatment of relapsed/refractory AML. Achieving remission depends on neither the risk group nor the time of relapse occurrence. The blast cell count in BM on the 14&lt;sup&gt;th&lt;/sup&gt;-16&lt;sup&gt;th&lt;/sup&gt; day of FLAG/FLAG-Ida treatment is a prognostic factor determining achievement and duration of remission. WT1 expression level in the early post-induction period is a sensitive DFS marker. MRD status and molecular genetic risk (ELN) group affiliation are essential prognostic factors determining DFS and OS.

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.

Clinical implication of minimal residue disease monitoring by WT1 gene detection and flow cytometry in myelodysplastic syndrome with allogeneic stem cell transplantation

目的探讨骨髓增生异常综合征（MDS）患者在接受异基因造血干细胞移植（allo-HSCT）后基于WT1基因及流式细胞术（FCM）监测微小残留病（MRD）的意义。方法分别采用实时定量聚合酶链反应（RQ-PCR）和8色FCM检测2011年2月至2015年10月于北京大学人民医院接受allo-HSCT的MDS患者移植前及移植后骨髓中WT1基因表达及异常免疫表型，并与临床结果进行相关分析。结果92例患者中，40例（48.2%）移植后WT1阳性，9例（10.8%）FCM阳性（FCM检测骨髓表型异常髓系幼稚细胞≥0.01%），27例（29.3%）患者MRDco阳性[连续2次WT1≥0.6%（间隔2周）或连续2次FCM阳性（间隔2周）或同一骨髓标本WT1≥0.6%且FCM阳性]。单因素分析显示移植后FCM阳性、MRDco阳性与复发相关（P<0.001，P=0.017）。FCM（+）、FCM（−）组移植后2年累积复发率（CIR）分别为66.7%、1.2%（P<0.001）；MRDco（+）、MRDco（−）组移植后2年CIR分别为23.0%、1.6%（P=0.004）。移植后WT1、FCM、MRDco预测复发的敏感性均为66.7%，特异性分别为59.0%、96.4%、74.7%。结论FCM和基于WT1/FCM的MRDco可作为MDS患者MRD监测的有效指标；基于MRDco标准进行干预可降低移植后复发率。

Gene mutation analysis in 12 Chinese children with congenital nephrotic syndrome

BackgroundCongenital nephrotic syndrome (CNS) is characterised by increased proteinuria, hypoproteinemia, and edema beginning in the first 3 months of life. Recently, molecular genetic studies have identified several genes involved in the pathogenesis of CNS. A systematic investigation of the genes for CNS in China has never been performed; therefore, we conducted a mutational analysis in 12 children with CNS,with the children coming from 10 provinces and autonomous regions in China.MethodsTwelve children with CNS were enrolled from 2009 to 2016. A mutational analysis was performed in six children by Sanger sequencing in eight genes (NPHS1, NPHS2, PLCE1, WT1, LAMB2, LMXIB, COQ6 and COQ2) before 2014, and whole-exome sequencing was used from 2014 to 2016 in another six children. Significant variants that were detected by next generation sequencing were confirmed by conventional Sanger sequencing in the patients’ families.ResultsOf the 12 children, eight patients had a compound heterozygous NPHS1 mutation, one patient had a de novo mutation in the WT1 gene, and another patient with extrarenal symptoms had a homozygous mutation in the COQ6 gene. No mutations were detected in genes NPHS2, PLCE1, LAMB2, LMXIB, and COQ2 in the 12 patients.ConclusionsThis study demonstrates that the majority of CNS cases (67%, 8/12 patients) are caused by genetic defects, and the NPHS1 mutation is the most common cause of CNS in Chinese patients. A mutational analysis of NPHS1 should be recommended in Chinese patients with CNS in all exons of NPHS1 and in the intron-exon boundaries.

Relationship between Quantitative Monitoring of WT1 Gene and Prognosis in Patients with Acute Leukemia

To investigate the exprassion of WT1 gene in patients with adult acute myeloid leukemia (AML) and its clinical significance. Sixty-three newly diagnosed patients with acute myeloid leukemia were selected. Quantitative RT-PCR was used to detect the expression of WT1 gene in the 63 AML patients and 20 non-AML controls. WT1 gene was highly expressed in AML patents and its expression in the low-risk group was significantly lower than that in middle-risk group and high-risk group (P<0.05), and no significant difference of WT1 gene expression between middle-risk and high-risk group was observed. In the patients of middle-risk and high-risk patients, the expression of WT1 gene in the remission group was significantly lower than that in the patients of non-remission after treatment (P<0.05). The non-remission patients after first treatment in middle-risk and high-risk group were treated with second induction therapy. After second induction therapy, the WT1 expression in remission patients was significantly decreased (P<0.05) in comparison with that in patients still in non-remission. There was a negative correlation between WT1 expression and the 2-year overall survival rate in the newly diagnosed middle and high-risk AML patients. The detection of WT1 gene expression can help to divide AML patients into low-/middle-/high-risk groups and to evaluate therapeutic response and clinical prognosis in middle and high-risk AML patients.