WT1 Mutations Research Articles

RAS Mutations Are Independently Associated with Decreased Overall Survival and Event-Free Survival in Patients with AML Receiving Induction Chemotherapy

Cai: Imago Biosciences, Inc.: Consultancy. Viny:Hematology News: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Other: Sponsored travel. Goldberg:American Society of Clinical Oncology: Research Funding; Abbvie: Research Funding; ADC Therapeutics: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Celgene: Consultancy. Tallman:BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stein:Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

Emerging Mutations at Relapse in Patients with FLT3-Mutated Relapsed/Refractory Acute Myeloid Leukemia Who Received Gilteritinib Therapy in the Phase 3 Admiral Trial

Smith: Revolution Medicines: Research Funding; Astellas Pharma: Research Funding; fujiFilm: Research Funding; Abbvie: Research Funding. Levis:Astellas: Consultancy, Research Funding; FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Daiichi Sankyo Inc: Consultancy, Honoraria. Perl:Astellas: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of trial data., Research Funding; BioMed Valley Discoveries: Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Other, Research Funding; Arog: Consultancy, Other: Non-financial support included travel costs for advisory board meetings.; AbbVie: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Actinium Pharmaceuticals: Consultancy, Honoraria, Other: Clinical Advisory Board member, Research Funding; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Non-financial support included travel costs for advisory board meetings.; Jazz: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; NewLink Genetics: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Takeda: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Bayer: Research Funding; FujiFilm: Research Funding; Novartis: Honoraria, Other: Advisory board, Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of the data., Research Funding. Martinelli:Daiichi Sankyo: Consultancy, Honoraria; Roche: Consultancy, Other: trial grant; Ariad: Consultancy, Other: trial grant; Janssen: Consultancy, Other: trial grant; Amgen: Consultancy, Other: trial grant; Pfizer: Consultancy, Other: trial grant; Abbvie: Consultancy, Honoraria, Other: trial grant; Celgene: Consultancy, Honoraria, Other: trial grant; Novartis: Consultancy, Other: trial grant; Incyte: Consultancy, Other: trial grant. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Montesinos:Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: Research support; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Abbvie: Research Funding; Astellas: Research Funding; Al Therapeutics: Research Funding; Forma: Research Funding; Incyte: Research Funding; Kite: Research Funding; Takeda: Research Funding. Larson:Celgene: Consultancy; Agios: Consultancy; Novartis: Honoraria, Other: Contracts for clinical trials. Yokoyama:Astellas: Other: Travel expenses. Recher:Incyte: Honoraria; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Yoon:MSD: Consultancy; Kyowa Hako Kirin: Research Funding; Janssen: Consultancy; Genentech, Inc.: Research Funding; Amgen: Consultancy, Honoraria; Yuhan Pharma: Research Funding; Novartis: Consultancy, Honoraria. Hill:Astellas: Employment; Ligacept, LLC.: Other: Stock, Patents & Royalties. Rosales:Astellas: Employment. Bahceci:Astellas: Employment, Patents & Royalties.

Genetic Characterization and Prognostic Relevance of Acquired Uniparental Disomies in Cytogenetically Normal Acute Myeloid Leukemia.

Uniparental disomy (UPD) is a way cancer cells duplicate a mutated gene, causing loss of heterozygosity (LOH). Patients with cytogenetically normal acute myeloid leukemia (CN-AML) do not have microscopically detectable chromosome abnormalities, but can harbor UPDs. We examined the prognostic significance of UPDs and frequency of LOH in patients with CN-AML.Experimental Design: We examined the frequency and prognostic significance of UPDs in a set of 425 adult patients with de novo CN-AML who were previously sequenced for 81 genes typically mutated in cancer. Associations of UPDs with outcome were analyzed in the 315 patients with CN-AML younger than 60 years. We detected 127 UPDs in 109 patients. Most UPDs were large and typically encompassed all or most of the affected chromosome arm. The most common UPDs occurred on chromosome arms 13q (7.5% of patients), 6p (2.8%), and 11p (2.8%). Many UPDs significantly cooccurred with mutations in genes they encompassed, including 13q UPD with FLT3-internal tandem duplication (FLT3-ITD; P < 0.001), and 11p UPD with WT1 mutations (P = 0.02). Among patients younger than 60 years, UPD of 11p was associated with longer overall survival (OS) and 13q UPD with shorter disease-free survival (DFS) and OS. In multivariable models that accounted for known prognostic markers, including FLT3-ITD and WT1 mutations, UPD of 13q maintained association with shorter DFS, and UPD of 11p maintained association with longer OS. LOH mediated by UPD is a recurrent feature of CN-AML. Detection of UPDs of 13q and 11p might be useful for genetic risk stratification of patients with CN-AML.

Uncovering the Genomic Landscape in Newly Diagnosed and Relapsed Pediatric Cytogenetically Normal FLT3-ITD AML.

Fms‐like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations, common in pediatric acute myeloid leukemia (AML), associate with early relapse and poor prognosis. Past studies have suggested additional cooperative mutations are required for leukemogenesis in FLT3‐ITD+ AML. Using RNA sequencing and a next‐generation targeted gene panel, we broadly characterize the co‐occurring genomic alterations in pediatric cytogenetically normal (CN) FLT3‐ITD+ AML to gain a deeper understanding of the clonal patterns and heterogeneity at diagnosis and relapse. We show that chimeric transcripts were present in 21 of 34 (62%) of de novo samples, 2 (6%) of these samples included a rare reoccurring fusion partner BCL11B. At diagnosis, the median number of mutations other than FLT3 per patient was 1 (range 0–3), which involved 8 gene pathways; WT1 and NPM1 mutations were frequently observed (35% and 24%, respectively). Fusion transcripts and high variant allele frequency (VAF) mutants, which included WT1,NPM1,SMARCA2,RAD21, and TYK2, were retained from diagnosis to relapse. We did observe reduction in VAF of simple or single mutation clones, but VAFs were preserved or expanded in more complex clones with multiple mutations. Our data provide the first insight into the genomic complexity of pediatric CN FLT3‐ITD+ AML and could help stratify future targeted treatment strategies.

Clinical and biological features of PTPN2-deleted adult and pediatric T-cell acute lymphoblastic leukemia

AbstractProtein tyrosine phosphatase nonreceptor type 2 (PTPN2) is a phosphatase known to be a tumor suppressor gene in T-cell acute lymphoblastic leukemia (T-ALL). Because the full clinicobiologic characteristics of PTPN2 loss remain poorly reported, we aimed to provide a comprehensive analysis of PTPN2 deletions within a cohort of 430 patients, including 216 adults and 214 children treated according to the GRAALL03/05 (#NCT00222027 and #NCT00327678) and the FRALLE2000 protocols, respectively. We used multiplex ligation-dependent probe amplification to identify an 8% incidence of PTPN2 deletion, which was comparable in adult (9%) and pediatric (6%) populations. PTPN2 deletions were significantly associated with an αβ lineage and TLX1 deregulation. Analysis of the mutational genotype of adult T-ALL revealed a positive correlation between PTPN2 deletions and gain-of-function alterations in the IL7R/JAK-STAT signaling pathway as well as PHF6 and WT1 mutations. Of note, PTPN2 and PTEN (phosphatase and tensin homolog) deletions were mutually exclusive. Regarding treatment response, PTPN2-deleted T-ALLs were associated with a higher glucocorticoid response and a trend for improved survival in children, but not in adults, with a 5-year cumulative incidence of relapse of 8% for PTPN2-deleted pediatric cases vs 26% (P = .177).

MON-309 THE NEW ZEALAND PAEDIATRIC EXPERIENCE OF DIFFUSE MESANGIAL SCLEROSIS 1991-2018

Diffuse mesangial sclerosis (DMS) is a rare cause of nephrotic syndrome in the infantile and childhood period. It is generally characterized by rapid progression to end-stage renal disease and can present in isolation or in association with Denys-Drash syndrome. The largest published series to date includes 14 children from a Spanish cohort 1973-2008. We describe the clinical presentation and outcome for our case series of children with DMS. We retrospectively reviewed all cases of DMS diagnosed via renal biopsy in our tertiary paediatric centre between 1991-2018. Starship Children’s hospital is the sole paediatric nephrology referral centre for New Zealand. A total of seven cases were identified consisting of four males and three females. Fiver patients were under 1 year of age at presentation. Two patients in the cohort were siblings. Three children had clinical evidence of Denys-Drash syndrome. The most recent five patients (presenting after year 2000) underwent genetic testing - three displayed WT1 mutations, including one patient who was found to have a novel mutation. This particular patient had a strong family history of focal segmental glomerulosclerosis. Unlike other published series, only four of the seven patients presented in end stage renal disease (ESRD). The remaining cohort displayed variable degrees of proteinuria only. Two patients with proteinuria underwent biopsy due to a significant family history of nephrotic syndrome. All four patients with ESRD on presentation required dialysis, and half of these patients subsequently had bilateral nephrectomies. Two patients proceeded to transplant with no relapse of the disease, though one transplant failed after fourteen years with the patient returning to haemodialysis. One patient died whilst on the deceased donor list due to cardiovascular complications. One patient is currently being worked up for living donor transplant. Two patients with proteinuria are currently being treated with ACE inhibitor therapy with improved proteinuria, normalisation of hypoalbuminaemia and maintainenance of a normal estimated GFR. One patient has moved overseas and is lost to follow up. There is a broad spectrum of DMS presentations ranging from mild proteinuria to ESRD. Karyotyping and genetic testing are important to establish the presence of syndromic associations and in particular WT1 mutations, as these patients require Wilm’s tumour surveillance if nephrectomies are not performed. Early detection of DMS and initiation of anti-proteinuric therapy is hoped to be beneficial to prolong native kidney function. Unfortunately, this may only be possible in patients for whom DMS is already suspected as a potential diagnosis due to a family history.

Abstract 2870: Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia

Abstract Survival for children with acute myeloid leukemia (AML) remains low, with a primary induction remission failure rate of 10-15%. Although some mutations have been identified that are associated with increased relapse risk, the genomic landscape of pediatric AML resistant to primary chemotherapy is not well characterized. As part of the TARGET initiative (Therapeutically Applicable Research To Generate Effective Treatments), we assembled a cohort of 28 pediatric AML patients with primary chemotherapy resistance and failure of induction chemotherapy, uniformly treated as part of the COG AAML0531 study. We analyzed whole-genome DNA, mRNA, and miRNA sequence data obtained at diagnosis and after induction chemotherapy. At least three genetically distinct groups were apparent in this cohort, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of NUP98 variants, and additional recurrent variants including those in KMT2C and MLLT10. Comparisons of mutations before and after chemotherapy revealed both common and distinct gene expression programs. On chemotherapy exposure, these leukemias exhibited diverse forms of clonal evolution. We observed selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination or contraction of FLT3, PTPN11, and NRAS mutant clones. These findings suggest diverse genetic mechanisms for primary chemotherapy resistance in pediatric AML, which should guide new approaches for its diagnosis and therapy. Citation Format: Nicole McNeer, John Philip, Heather Geiger, Rhonda E. Ries, Vincent-Philippe Lavallee, Michael Walsh, Minita Shah, Kanika Arora, Anne-Katrin Emde, Nicolas Robine, Todd A. Alonzo, E. Anders Kolb, Alan S. Gamis, Malcom Smith, Daniela Se Gerhard, Jaime Guidry-Auvil, Soheil Meshinchi, Alex Kentsis. Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2870.

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.

S841 DYNAMIC GENETIC EVOLUTION IN MYELODYSPLASTIC SYNDROME AND THE CLINICAL IMPLICATIONS

Background:Myelodysplastic syndromes (MDS) are heterogeneous clonal myeloid disorders with increased risk of transformation to acute myeloid leukemia (AML). To understand the role of genetic evolution in the progression of MDS and its prognostic implication, comprehensive mutation analyses of large MDS cohorts sequentially by next generation sequencing during the clinical course are mandatory.Aims:The study was aimed to explore the evolution profile of genetic alterations and its clinic‐prognostic impact in MDS patients.Methods:Totally, 161 de novo MDS patients diagnosed according to the 2016 WHO classification who had paired samples both at diagnosis and subsequent follow‐ups were recruited. Among them, 81 patients had disease progression: 24 patients progressed to high‐risk MDS and 57 progressed to AML. Gene mutations were analyzed by targeted next generation sequencing of 54 genes involving in myeloid malignancies using TruSight Myeloid Panel.Results:The most prevalent gene mutations at diagnosis and progression were similar, including ASXL1, followed by RUNX1, SRSF2, and U2AF1 mutations. The most common genetic evolution was acquisition of NRAS mutation (16.0%), followed by that of chromosome 8 abnormalities (14.8%), and RUNX1 mutations (13.5%). Among the 81 patients with disease progression, 57 (70.4%) patients had genetic evolution: 18 patients had only cytogenetic evolution, 22 had only mutation shift (either acquisition or loss), and 17 had both cytogenetic evolution and mutation shift. The patients with disease progression had a higher frequency of mutation shift than those with stable disease (48.1% vs. 30.0%, P = 0.024). The gene mutation numbers were not associated with the risk of disease at diagnosis, but at progression, patients with acute myeloid leukemia (AML) had more gene mutation numbers than those with high risk MDS. Progression to AML rather than to high‐risk MDS and presence of cytogenetic or genetic evolution predicted poorer overall survival (OS) after progression (Fig 1A), irrespective of the original subtypes of MDS.We used SciClone to analyze the clonal architecture of gene mutations at diagnosis and at disease progression and identified two clusters of gene mutations. Cluster 1 gene mutations, including DNMT3A, NRAS, RUNX1, WT1, IDH2, splicing factors (including SRSF2, SF3B1, and U2AF1), and TET2 mutations, tended to have increasing allelic burdens or be acquired at progression; cluster 2 gene mutations, including ASXL1, SETBP1, STAG2, BCOR, and TP53 mutations, tended to be stationary, decreasing, or lost at disease progression. At diagnosis, the cluster 1 gene mutations were significantly more prevalent in MDS with subsequent progression to secondary AML but the cluster 2 gene mutations were more common in those with progression to high‐risk MDS. We risk‐stratified patients into three groups at diagnosis: the group 1 patients with cluster 1 ± cluster 2 gene mutations, the group 2 patients with the only cluster 2 gene mutations, and the group 3 patients with neither cluster 1 nor cluster 2 gene mutations. The group 3 patients had the longest OS, followed by the group 1, and the group 2 patients had the worst OS (Fig 1B).Summary/Conclusion:More than two‐thirds of MDS patients experienced genetic evolution during disease progression and the presence of the genetic evolution had a poor prognostic impact. It is necessary to develop novel treatment strategies for patients with genetic evolution in the near future.image

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.

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

Genetic Testing Proves Crucial in Case of Ambiguous Genitalia and Renal Masses

ObjectiveThe Denys-Drash syndrome consists of a triad of ambiguous genitalia, Wilm's tumor and nephrotic syndrome. MethodsWe present a diagnostically challenging case of an XY patient with female appearance and Müllerian structures with a WT1 mutation. ResultsThese genetic findings resulted in gonadal dysgenesis, end-stage renal disease, and precursor changes to Wilm's tumor in both kidneys. Genetic testing proved critical in this case, helping to solidify a diagnosis and guiding our decision to proceed with bilateral nephrectomy and bilateral gonadectomy. ConclusionsDenys-Drash syndrome can present quite dramatically. WT1 testing should be considered early in the workup for patients with differences of sexual development, particularly those with 46XY karyotype.

Abstract P3-06-01: Clonal evolution and heterogeneity in breast tumors treated with neoadjuvant HER2-targeted therapy

Abstract Background: Understanding to what extent a breast tumor's genetic composition may change over the course of a few months of neoadjuvant therapy has implications for optimal therapeutic approach. However, genomic changes observed across treatment may result from either treatment-induced clonal evolution or geographically disparate sampling of a heterogeneous tumor. We sought to characterize the geographic heterogeneity in primary breast tumors, and to incorporate this information into analysis of clonal evolution with neoadjuvant therapy. Methods: We assembled the largest cohort to date of multi-region (n=2-3) whole-exome sequenced (WES) or whole-genome sequenced untreated primary breast tumors with matched normal and adequate tumor purity for analysis: four tumors with data generated for this study and five tumors compiled from three previous studies. We also generated the first cohort of multi-region (n=2-6) WES breast tumors post-neoadjuvant HER2-targeted therapy and chemotherapy, sequencing one region from a pre-treatment diagnostic specimen, multiple regions from the post-treatment surgical specimen, and matched normal for five HER2+ breast tumors that did not achieve a pathologic complete response. We used an agent-based model of spatial tumor growth to investigate whether the mutational patterns we observed with treatment were consistent with pre-existing heterogeneity or treatment-induced selection. Results: In untreated primary breast tumors, on average 30% (range 1-70%) of apparently clonal mutations from a single region were absent or rare in a second, spatially disparate region (high-frequency regional, or HFR). Intra-tumor heterogeneity was similar post-treatment (HFR 28%, range 10-54%), and was higher in breast tumors than in previously analyzed colon, brain, lung, and esophageal tumors. Simulation studies confirmed that with high heterogeneity as observed in breast tumors, analysis of one pre-treatment and one post-treatment region could not distinguish treatment-induced clonal evolution from pre-existing heterogeneity; however, obtaining at least two post-treatment regions allowed for detection of clonal shifts with treatment. Analysis of multi-region data revealed that clonal replacement occurred with neoadjuvant therapy in two of the five tumors. Candidate causes of therapeutic resistance included amplifications in CCND1, ERBB4, and MYC in one subclone, and functional protein-altering mutations in ERCC2, SMO, and WT1 in another. Mathematical modeling suggested that these putative resistant subclones comprised 0.02-12.5% of the overall pre-treatment cell population, substantially larger than previous estimates of resistant tumor clone size. Conclusions: WES data from multiple regions of untreated and treated primary breast tumors revealed considerable heterogeneity that remained present throughout treatment with chemotherapy and HER2-targeted therapy, even while major clonal sweeps took place in a minority of tumors. Obtaining at least two samples for analysis from breast tumors post-neoadjuvant therapy may reveal the tumor's evolutionary path and, especially as increasing numbers of molecular and immune therapeutic targets are identified, inform new clinical strategies. Citation Format: Caswell-Jin JL, McNamara K, Reiter JG, Sun R, Hu Z, Ma Z, Suarez CJ, Tilk S, Raghavendra A, Forte V, Chin S-F, Bardwell H, Provenzano E, Caldas C, Lang J, West R, Tripathy D, Press MF, Curtis C. Clonal evolution and heterogeneity in breast tumors treated with neoadjuvant HER2-targeted therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-01.

Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia.

Acute myeloid leukemias (AML) are characterized by mutations of tumor suppressor and oncogenes, involving distinct genes in adults and children. While certain mutations have been associated with the increased risk of AML relapse, the genomic landscape of primary chemotherapy resistant AML is not well defined. As part of the TARGET initiative, we performed whole-genome DNA and transcriptome RNA and miRNA sequencing analysis of pediatric AML with failure of induction chemotherapy. We identified at least three genetic groups of patients with induction failure, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of apparent NUP98 mutations, and additional recurrent variants including those in KMT2C and MLLT10. Comparison of specimens before and after chemotherapy revealed distinct and invariant gene expression programs. While exhibiting overt therapy resistance, these leukemias nonetheless showed diverse forms of clonal evolution upon chemotherapy exposure. This included selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination of FLT3, PTPN11, and NRAS mutant clones. These findings delineate genetic mechanisms of primary chemotherapy resistance in pediatric AML, which should inform improved approaches for its diagnosis and therapy.

Genetics of congenital and infantile nephrotic syndrome.

Congenital and infantile nephrotic syndrome (CNS and INS) are rare inherited defects in glomerular filtration involving a variety of gene mutations. This study aimed to analyze all genetic mutations associated with congenital and infantile nephrotic syndrome treated at our institution. We also discussed our different approach secondary to culture and resources. A retrospective single-center study of all children diagnosed as NS before the age of 1 year over a duration of over one decade. Twenty-nine children (12 boys) were included in the study. Their median age (range) was 2.4 (0.1-12) months (20 CNS and 9 INS). Consanguinity was present in 90% of children. The genetic analysis' results were only available for 20 children. An underlying causative homozygous mutation was detected in 18 children (90%): NPHS1 (9), NPHS2(2), LAMB2(3), PLCE1(1), WT1(1), and ITSN1 novel mutation (2). One child had heterozygous mutation of NPHS2 and another child had heterozygous mutation of NPHS1 which could not explain the disease. All CNS cases were all managed with intermittent intravenous albumin infusion, ACEi, diuretics, and indomethacin. None of the children were managed by nephrectomy followed by peritoneal dialysis (PD) because of limited resources. Only one child achieved partial remission, while 15 children died at a median (range) age of 5.8 (1.25-29) months. The remaining 14 children were followed up for an average of 36 (3.9-120) months. Three children progressed to end-stage kidney disease and PD was performed in only two children. NPHS1 is the main underlying cause of CNS and INS in our study population. CNS and INS were associated with high morbidity and mortality.

Allogenic Hematopoietic Cell Transplantation (HCT) for Chronic Myelomonocytic Leukemia: Clinical, Cytogenetic, and Mutational Risk Factors Associated with Survival

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy characterized by persistent monocytosis, combined with myeloid proliferation and dysplasia. Here, we report HCT outcomes in 129 patients (pts) with CMML, 7-74 (median 55) years of age, with follow-up extending to 25 years. Patients were conditioned with various intensity regimens and transplanted from related (N=45) or unrelated donors (N= 84). The source of stem cells was marrow in 34, mobilized peripheral blood cells in 93, and cord blood in 2 patients. The HCT-CI was 0-11 (median 3). Somatic mutations present pre-HCT were analyzed with a mutation panel sequencing for 75 genes. Fig. 1A and 1B show overall survival by WHO disease criteria and the Spanish cytogenetic classification, respectively. Acute graft-versus-host disease (GVHD) grades II-IV occurred in 74% and chronic GVHD in 43%. Relapse incidence was 32%. Overall and progression-free survival at 10 years was 28% and 29%, respectively. Multivariate regression models showed the following: Blast count >20% at HCT (CMML-T: HR 1.67, CI 0.9-3.2, p=0.13, Fig. 1A), high-risk cytogenetics (HR 1.88, CI 1.2-3.0, p=0.01, Fig. 1B) and high HCT comorbidity index (HCT-CI≥4: HR 1.99, CI 1.2-3.4, p=0.01, Fig. 1 C) negatively impacted survival. There were 52% and 42% of patients who carried mutations in ASXL1 and TET2 and they were not significantly associated with inferior survival (ASXL1: HR=1.6, CI 0.3-7.6, p=0.54, TET2: HR=1.3, CI 0.4-3.9, p=0.63). Mutations in ATRX (HR=17.3, CI 4.1-73, p=0.0005) and WT1 (HR=6.3 CI 1.6-24, p=0.01), however, were associated with inferior survival. The total number of mutations (> 10 mutations, HR=3.5, CI 1.4-7.3, p=0.02) and more mutations in genes regulating epigenetic processes (HR 1.8, CI 1.1-2.8, p=0.01) were associated with relapse. The molecular CMML-specific prognostic scoring system (CPSS-Mol, Elena et al. 2016) was not correlated with relapse or survival. Unsupervised clustering of the correlation matrix revealed distinct associations between mutations and clinical features (Fig. 2), Group 1, proliferative type (mutations in mitotic signaling pathways associated with high WBC/blast counts and high risk disease by prognostic scoring systems), and Group 2, dysplastic type (mutations in genes regulating epigenetic processes including ATRX and WT), which was associated with unfavorable outcome. In summary, a proportion of patients with CMML achieve lasting remissions following HCT. However, relapse and overall mortality remain high. Molecular annotation uncovered distinct subgroups of CMML, and higher number of mutations, in particular, of epigenetic regulators, may confer unfavorable transplant outcomes. Incorporation of mutations in addition to cytogenetics and comorbidity scores should improve transplant prognostication.

Next generation sequencing (NGS) to improve the diagnosis and management of patients with disorders of sex development (DSD).

Disorders of sex development (DSDs) are a diverse group of conditions where the chromosomal, gonadal or anatomical sex can be atypical. The highly heterogeneous nature of this group of conditions often makes determining a genetic diagnosis challenging. Prior to next generation sequencing (NGS) technologies, genetic diagnostic tests were only available for a few of the many DSD-associated genes, which consequently had to be tested sequentially. Genetic testing is key in establishing the diagnosis, allowing for personalised management of these patients. Pinpointing the molecular cause of a patient’s DSD can significantly impact patient management by informing future development needs, altering management strategies and identifying correct inheritance pattern when counselling family members. We have developed a 30-gene NGS panel, designed to be used as a frontline test for all suspected cases of DSD (both 46,XX and 46,XY cases). We have confirmed a diagnosis in 25 of the 80 patients tested to date. Confirmed diagnoses were linked to mutations in AMH, AMHR2, AR, HSD17B3, HSD3B2, MAMLD1, NR5A1, SRD5A2 and WT1 which have resulted in changes to patient management. The minimum diagnostic yield for patients with 46,XY DSD is 25/73. In 34/80 patients, only benign or likely benign variants were identified, and in 21/80 patients only variants of uncertain significance (VOUS) were identified, resulting in a diagnosis not being confirmed in these individuals. Our data support previous studies that an NGS panel approach is a clinically useful and cost-effective frontline test for patients with DSDs.

Once-Daily Low-Dose Cyclosporine A Treatment with Angiotensin Blockade for Long-Term Remission of Nephropathy in Frasier Syndrome

Cyclosporine A is known to be effective in some genetic podocyte injury. However, the efficacy of cyclosporine A depends on the degree of histopathological findings, and the relationship between long-term use and renal prognosis remains unknown. Frasier syndrome is a rare genetic disorder caused by intronic mutations in WT1, and is characterized by progressive glomerulopathy, a 46,XY disorder of sex development, and an increased risk of gonadoblastoma. We report here a 16-year-old phenotypically female patient with Frasier syndrome. A renal biopsy at the age of seven years showed segmentally effaced podocyte foot processes with no evidence of glomerulosclerosis. Steroid-resistant proteinuria progressed to the nephrotic range at the age of 10 years, which responded to once-daily administration of cyclosporine A with low two-hour post-dose cyclosporine A (C2) levels; she then achieved stable partial remission in combination with renin-angiotensin system (RAS) blockade. At the age of 12 years, examinations for delayed puberty confirmed the diagnosis of Frasier syndrome. The second renal biopsy showed widespread foot process effacement and a minor lesion of segmental glomerulosclerosis without findings suggestive of cyclosporine A nephropathy. She maintained partial remission and normal renal function with the continuation of once-daily low-dose cyclosporine A. The C2 levels required for the remission were between 212 and 520 ng/ml. Cyclosporine A dosages sufficient for maintaining the C2 levels were 1.1-1.2 mg/kg per day. In conclusion, the long-lasting treatment of once-daily low-dose cyclosporine A with RAS inhibition was effective for induction and maintenance of partial remission in Frasier syndrome.

Prognostic and Clinicopathological Significance of MiR-155 in Hematologic Malignancies: A Systematic Review and Meta-analysis.

Background: Aberrant miR-155 expression has been reported in various types of hematologic malignancies. However, the prognostic and clinicopathological value of miR-155 remains unclear. Here, we performed this systemic review and meta-analysis to comprehensively evaluate the prognostic and clinicopathological significance of miR-155 expression in hematologic malignancies.Methods: We systematically searched the PubMed, EMBASE, ISI Web of Science, Cochrane library databases and OVID to identify eligible studies published from Jan 1, 2008 to Aug 1, 2018. The pooled hazard ratios (HRs) and odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were used to detect the prognostic and clinicopathological role of miR-155 in hematologic malignancies.Results: A total of 18 studies including 2316 patients were enrolled in the present meta-analysis, indicating significant association between elevated miR-155 expression and poor overall survival (OS) in 2114 patients (pooled HR = 1.72, 95%CI [1.50-1.97], p<0.001). Elevated miR-155 expression level was related to shorter event free survival (EFS, pooled HR = 1.55, 95%CI [0.94-2.57], P=0.002), disease free survival (DFS, pooled HR = 1.38, 95%CI [1.13-1.68], P=0.001), progress free survival (PFS, pooled HR = 1.58, 95%CI [1.06-2.35], p<0.001) and treatment free survival (TFS, pooled HR = 1.67, 95%CI [1.16-2.39], P=0.006). Additionally, overexpression of miR-155 was found to be significantly related to FLT3/ITD presence (OR=4.751, 95%CI [3.229-6.990], P<0.001), more WT1 mutation (OR=2.090, 95%CI [1.240-3.522], P=0.006) and less CEBPA mutation (OR=0.477, 95%CI [0.286-0.794], P=0.004) in 552 AML patients.Conclusion: MiR-155 expression was found to be associated with several leukemia-related phenotype and poor prognosis in hematologic malignancies. Therefore, miR-155 overexpression might be a convinced unfavorable prognostic indicator that helps the clinical decision-making process.

Molecular Genetic Characterization of Acute Myeloid Leukemia With Trisomy 4 as the Sole Chromosome Abnormality.

The aim of the study was to determine the genetic and molecular consequences of trisomy 4, a recurrent but rare chromosomal abnormality in acute myeloid leukemia (AML). Interphase fluorescence in situ hybridization, reverse transcriptase-quantitative polymerase chain reaction for 28 chromosomal gene translocations/fusion genes, and targeted sequencing analyses were performed on five AMLs with trisomy 4 as the sole chromosomal anomaly. An NPM1 frameshift mutation was found in all leukemic bone marrows, DNMT3A, FLT3, and IDH1 mutations were found in three, KIT and NRAS mutations in two, whereas IDH2 (R140Q), RUNX1, and WT1 mutations were found in only one patient each. The three patients with a DNMT3A (R882H) mutation have died. In contrast, the two patients whose leukemic cells were without this mutation, are alive 55 and 31 months after diagnosis, respectively. The results suggest a possible association between trisomy 4 and additional mutations that may influence prognosis.