Conventional Karyotyping Research Articles

KMT2A Partial Tandem Duplications (KMT2A-PTD) Is a Rare, but Recurrent Genomic Event in Childhood AML and Associated with High Rate of Co-Occurring FLT3 Mutations

KMT2A partial tandem duplication (KMT2A-PTD), characterized by a large internal duplication spanning 6-8 exons, has been documented in adult AML with a prevalence of 3-10% and is associated with poor outcomes. Despite the high prevalence of KTM2A fusions and known associated outcomes, prevalence and clinical implications of KMT2A-PTD has not been well characterized in childhood AML. We interrogated the transcriptome and associated clinical and genomic data from pediatric AML patients treated on COG AAML1031 to define the prevalence of KMT2A-PTD, frequency of co-occurring genetic mutations and outcomes associated with this variant.Ribo-depleted RNA seq data from 1,294 patients with AML (age 0-29 years) enrolled on COG AAML1031 with available cytogenetic, molecular, and clinical data were utilized. Gene fusions, internal tandem duplications (ITD) and partial tandem duplications (PTD) were detected by Cicero. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytogenetic and molecular abnormalities. Response was measured by 5-year overall survival (OS), event-free survival (EFS), disease-free survival (DFS) and relapse risk (RR).Of the 1,294 patients studied, KMT2A-PTD was identified in 20 patients (1.5%). Evaluation of karyotype demonstrated striking paucity of karyotypic alterations with 75% with normal karyotype; 3 cases (15%) of trisomy 11 were identified. In contrast to KMT2A-rearranged (KMT2A-r) AML, which has the highest prevalence in young patients (median age 3.3 years), the median age with KMT2A-PTD was 13.4 years (p<0.0001). Further analysis demonstrated a high rate of co-occurrence of FLT3 mutations; 15 patients (75%) had FLT3-ITD (N=13; 65%) and/or FLT3 activating mutations (N=5; 25%). The rate of co-occurring FLT3-ITD was significantly higher in the KMT2A-PTD cohort compared to patients without KMT2A-PTD (65% vs. 19%, p<0.001). Prevalence of KMT2A-PTD in patients with FLT3-ITD was 5.7%. Additional co-occurring genetic mutations were identified in WT1 (20%) and the RAS pathway. There was a striking enrichment of mutations that are commonly associated with AML in older adults with 8/20 patients (40%) harboring mutations in IDH1/IDH2, U2AF1, and TP53, far in excess of what is seen in other pediatric AML patients (Figure 1A).We evaluated the significance of KMT2A-PTD on clinical outcome. Patients with KMT2A-PTD had a poor response to induction therapy with a morphologic CR rate of 45% and rate of residual disease (MRD) positivity by flow cytometry after initial course of therapy was 40%. Additionally, high rates of relapse were noted for patients with KMT2A-PTD with a RR of 53%. EFS for patients with and without KMT2A-PTD was 39% vs. 46%, respectively (p=0.54) with a corresponding OS of 58% and 64% (p=0.61). Outcome analysis for the KMT2A-PTD cohort demonstrated dual KMT2A-PTD/FLT3-ITD patients had an EFS of 46% compared to 29% for KMT2A-PTD/non-ITD patients (p=0.62, Figure 1B). We inquired whether hematopoietic stem cell transplant (HSCT) may modify outcomes in patients with KMT2A-PTD. Of the 20 patients with KMT2A-PTD, 7 (35%) proceeded to HSCT in CR1, with the indication in 6 of 7 patients due to FLT3-ITD high allelic ratio (HAR, ≥0.4). Stem cell transplant recipients had a DFS of 83% vs. 0% for those who continued on protocol chemotherapy (p=0.002, Figure 1C) with corresponding OS of 100% vs. 43%, respectively (p=0.05).In this large cohort of childhood AML patients treated on AAML1031, we identified a small subset of patients with KMT2A-PTD and show the high prevalence of co-occurring FLT3 mutations. Although KMT2A-PTD patients with and without FLT3-ITD had similar outcomes, the cohort who received HSCT in CR1 (most with FLT3-ITD) experienced improved outcomes compared to the rest of the cohort, suggesting that intensification of therapy may benefit this group of patients overall. Further research into KMT2A-PTD in pediatric AML will guide future risk-adapted therapy and enhance understanding of biologic implications of this lesion, including whether altered KMT2A may serve as a therapeutic target as it may for KMT2A-r AML. [Display omitted] DisclosuresPollard: Syndax: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees.

Non-Invasive Genetic Profiling and Monitoring in Myelodysplastic Syndromes

Introduction. Myelodysplastic syndromes (MDS) are clonal heterogeneous disorders in which molecular studies and cytogenetics are essential for diagnosis, classification and prognosis. Cell-free DNA (cfDNA) analysis has been reported as a reliable non-invasive approach for detecting molecular abnormalities in MDS. However, there is limited information about cytogenetic alterations and monitoring in cfDNA from MDS patients.Objective. To assess and monitor the molecular and cytogenetic abnormalities by next generation sequencing (NGS) using cfDNA from patients with MDS.Patients and methods. Bone marrow (BM) aspirates and peripheral blood (PB) samples were collected from 70 newly diagnosed or treatment-naïve patients with MDS (Table). PB samples from 21 healthy controls were also studied. Molecular characterization was performed in paired samples of BM DNA and cfDNA by NGS in all patients. Libraries were prepared using a custom panel including 48 myeloid-associated genes and genomic regions localized at the most frequently altered chromosomes in MDS (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina. 20/70 (28.6%) showed cytogenetic/FISH alterations at diagnosis, 2 of them with infrequent alterations in MDS, not covered by the panel (+14, del(9q)). 6 cases presented chromosome Y loss as a single alteration, not covered by the NGS panel. Copy number variant (CNV) analysis was performed by NGS to detect cytogenetic alterations in both cfDNA and BM, and confirmed by chromosomal microarrays (CMA) in BM DNA (CytoScan/OncoScan, ThermoFisher).Results. We obtained a median amount of cfDNA of 58.4 ng/ml in MDS patients that was significantly higher than that obtained in healthy controls (median 32.4 ng/ml, P=0.023). Sequencing of BM DNA and cfDNA showed a comparable mutational profile (187/201 mutations, 93.0% concordance). The most frequently mutated genes were TET2 (44.3%), SF3B1 (37.1%), ASXL1 (20.0%), DNMT3A (20.0%), SRSF2 (15.7%), ZRSR2 (12.9%) and U2AF1 (12.9%). A strong correlation was observed between the VAFs of BM DNA and cfDNA (r s=0.797, P<0.001). VAFs of SF3B1 mutations detected in cfDNA were significantly higher than VAFs in BM DNA (P=0.016).The analysis of cytogenetic alterations by NGS showed abnormalities in 10/70 MDS patients in both BM DNA and cfDNA. Interestingly, in a patient without analyzable metaphases in karyotype, del(20q) was found by NGS and confirmed by CMA. Overall, CMA and NGS were highly concordant to detect chromosomal aberrations although they do not reach the sensitivity achieved by karyotype/FISH (Figure 1). However, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA.Molecular and cytogenetic alterations were monitored in sequential samples from 7 cases (median follow up: 13 months, range 10-30). We observed an excellent correlation between the VAF of mutations in BM DNA and ctDNA across multiple matched time points. A decrease in the VAF was detected in patients responding to therapy (either hypomethylating agents or chemotherapy), but not in non-responding patients (Figure 2). Of note, cfDNA analysis also showed cytogenetic evolution in 2 cases not responding to azacitidine (del(12p) and +21). From the two untreated patients, one acquired a subclonal del(7q) not detected by NGS and observed only in few metaphases, and the second patient showed a clonal expansion of the NF1 mutation at the time of AML transformation.Conclusions. Analysis of cfDNA allows the characterization and monitoring of molecular abnormalities in patients with MDS. Cytogenetic alterations were detectable in most cases by NGS in both BM DNA and cfDNA although with a lower detection rate than karyotype/FISH.Acknowledgements. ISCIII-FEDER, PI16/0153, PI19/0005, 2017SGR205, PT20/00023 and XBTC.Figure/Table legends:Table. MDS patients included, classified by disease subtype. (SLD: single lineage dysplasia; MLD: multilineage dysplasia; RS: ring sideroblasts; EB: excess blasts; MDS-U: MDS-unclassifiable)Figure 1. Detection of cytogenetic alterations by conventional karyotype, FISH, CMA and NGS.Figure 2. Monitoring of molecular and cytogenetic alterations in 7 patients with MDS. 5 patients receiving treatment (3 azacitidine, 1 FLAG-IDA + HCT, 1 inhibitor of hypoxia-inducible factor (HIF)) and 2 untreated cases were included. BM VAF dynamics are shown with a dotted line and cfDNA dynamics are shown with a solid line. [Display omitted] DisclosuresBesses: Gilead: Research Funding. Salar: Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Gilead: Research Funding; Celgene: Consultancy, Speakers Bureau. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

Progression-Free Survival (PFS) According to the Presence of Adverse Cytogenetic Abnormalities in Patients (pts) with Multiple Myeloma (MM) Receiving Ixazomib (ixa)-Based vs Placebo (pbo)-Based Therapy: A Pooled Analysis of the TOURMALINE-MM1, MM2, MM3, and MM4 Phase 3 Studies

Progression-Free Survival (PFS) According to the Presence of Adverse Cytogenetic Abnormalities in Patients (pts) with Multiple Myeloma (MM) Receiving Ixazomib (ixa)-Based vs Placebo (pbo)-Based Therapy: A Pooled Analysis of the TOURMALINE-MM1, MM2, MM3, and MM4 Phase 3 Studies

Oligoblastic (

Introduction: The current World Health Organization (WHO) classification considers acute myeloid leukemia (AML) with PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 to be AML regardless of blast count. Myeloid neoplasms with KMT2A rearrangement present with high blast percentages and are classified as AML in the vast majority of cases; however, rare myeloid neoplasms with KMT2A rearrangement may present with <20% blasts and these are currently considered to be myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMML). We questioned whether these oligoblastic KMT2A rearranged neoplasms shared similar biology with AML, and if this genetic finding should be considered to be AML-defining.Methods: We searched the archives of three large institutions in the United States for untreated adult with oligoblastic (<20%) myeloid neoplasms with KMT2A rearrangement and untreated adult AML patients with KMT2A rearrangement and compared their clinical and laboratory characteristics. KMT2A rearrangement was detected by chromosomal analysis and/or fluorescence in-situ hybridization (FISH) using KMT2A breakapart probe. KMT2A rearrangement partners were assessed based on a conventional karyotype results and/or RNA based fusion assay.Results: Between 2010 and 2021 we identified 22 patients with MDS or CMML and KMT2A rearrangement and 88 patients with AML and KMT2A rearrangement. The clinical and laboratory information are summarized below. MDS/CMML with KMT2A-rearrangement showed a similar age and sex distribution to AML, but had more patients with preceding history of chemotherapy for unrelated malignancies (68% vs 37%, p=0.012). Aside from a low blast count in PB and BM, patients with MDS/CMML had a lower WBC and were less anemic and thrombocytopenic. MDS/CMML patients also showed a lower percentage of BM cells with KMT2A rearrangement by FISH (58% vs. 92%, p<0.0001), and significantly less likely to have MLLT3 as the partner gene (23% vs. 58%, p=0.0005). With a similar mutation frequency among patients tested (42% versus 34%, p=0.22), MDS/CMML patients had significantly fewer mutations involving the RAS pathway (KRAS, NRAS, FLT3, PTPN11) than AML patients (p=0.0035).Seventeen of 22 MDS/CMML patients (77%) progressed to AML with a median time of progression being 4 months (range, 1-27 months). Of the 5 patients who did not progress to AML, 3 received allogeneic stem cell transplantation (SCT) and remain in complete remission, while 2 remain AML-free 3 and 16 months after the initial diagnosis.With a median follow-up of 12 months (range, <1-117), 15 MDS/CMML patients and 58 AML patients died. There was no difference in use of SCT to treat MDS/CMML versus AML (p=0.47). MDS/CMML and AML patients displayed similar overall survival (OS) (p=0.9059, Figure 1). There was also no statistical difference in OS between MDS/CMML and AML when therapy-related (p=0.569) and de novo (p=0.962) neoplasms were analyzed separately. On multivariable analysis, therapy-related disease (HR 1.774 [1.058-2.975], p=0.03), SCT (HR 0.166 [0.000-0.291], p<0.0001), and platelet count (HR 0.992 [0.000-0.998], p=0.013) were independently associated with shorter OS. Neither AML versus MDS/CMML nor blast percentage (whether dichotomized as <5% or >=5% or as a continuous variable) were associated with shorter OS.Conclusion: Oligoblastic myeloid neoplasms with KMT2A rearrangement tend to progress rapidly to overt AML and demonstrate similar aggressive behavior to AML patients with KMT2A rearrangement, irrespective of therapy relatedness. Patients with KMT2A rearrangement and <20% blasts are currently classified as MDS or CMML, and may be inadequately treated. Our findings suggest that all myeloid neoplasms with KMT2A (MLL) rearrangement should be classified as AML, irrespective of the blast count. [Display omitted] DisclosuresRavandi: AbbVie: Honoraria, Research Funding; Prelude: Research Funding; Taiho: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Issa: Novartis: Consultancy, Research Funding; Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding. Pozdnyakova: Scopio Labs: Consultancy. Wang: Stemline Therapeutics: Honoraria.

Conventional Cytogenetic and Molecular Analysis in Acute Myeloid Leukemia (AML) and Their Association with Overall Survival

Abstract Background: Conventional cytogenetic is one of the most important diagnostic tools for predicting the overall survival of the patients. Molecular genetics in acute myeloid leukemia (AML) has provided insights into the molecular mechanism of leukemogenesis. In this study we aimed to investigate the impact of cytogenetic and molecular methods on the survival of patients with de novo established AML in order to achieve a useful marker or test in the process of predicting the disease course. Material and methods: Eighty newly diagnosed AML patients who were treatment naive entered the study. Cytogenetic and molecular studies such as, the conventional karyotyping, sequencing and reverse transcriptase real time quantitative PCR (RT-qPCR) were included. Overall survival was calculated by Kaplan-Maier technique and the data were analyzed by SPSS.V.19. Results: Among 80 patients, 36 (45%) were female and 44 (55%) were male patients. Patients’ median age was 29 years, ranging from 1 to 76 years. The mean overall survival was 19 months (95% CI: 1523 months). The 1-year AML survival rate was 61%. There were significant differences in overall survival between the NPM1-mutated groups compared to the patients without any mutations (19% versus 61%) (p &lt; 0.032). Conclusion: This study makes a significant contribution in assessing the prognostic value of cytogenetic and molecular markers. This study showed the heterogeneity of de novo AML that involved various factors and prevalence of distinct cytogenetic subgroups. Our data in comparison with other population-based studies, confirmed a differential distribution of cytogenetic and molecular classification indicating geographic heterogeneity.

Comprehensive Assessment of Fetal Bilateral Ventriculomegaly Based on Genetic Disorders, Cytomegalovirus Infection, Extra Prenatal Imaging and Pregnancy Outcomes in a Tertiary Referral Center.

ObjectiveThis retrospective study aimed to systematically evaluate the genetic disorders, cytomegalovirus (CMV) infection, extra ultrasound findings and outcomes of fetuses with bilateral ventriculomegaly (BVM).MethodsData from pregnancies with fetal BVM were obtained between 2014 and 2020. The cases were divided into groups of isolated bilateral ventriculomegaly (IBVM) and non-isolated bilateral ventriculomegaly (NIBVM) according to the presence of extra prenatal imaging. Subgroups of mild, moderate, and severe were determined according to lateral ventricle widths. The NIBVM group was further classified into pregnancies with soft markers, non-structural abnormalities, and structural abnormalities.ResultsA total of 353 pregnancies were enrolled, including 153 cases of IBVM and 200 cases of NIBVM. Conventional karyotyping was performed on 192 samples, and 15 cases of numerical abnormalities and 3 cases of unbalanced structural abnormalities were identified. Chromosomal microarray analysis (CMA) was concurrently performed on 108 of them and revealed additional 5 cases (4.7%) of copy number variants with clinical significance. CMV DNA testing was performed on 154 of the 192 cases that underwent invasive prenatal diagnosis, and a positive result was found in 2 (1.3%) cases. In the IBVM group, the percentage of favorable prognosis in the mild, moderate and severe pregnancies were 94.4%, 79.2%, and 4.8%, respectively, and the termination of pregnancy (TOP) rates were 4.6%, 20.8%, and 85.7%, respectively. In both the mild and moderate NIBVM, the TOP rates progressively increased and the favorable prognosis survival rates progressively decreased relative to the soft markers, non-structural abnormalities, and structural abnormalities, respectively. Approximately 94.1% of severe NIBVM ended in termination.ConclusionGenetic disorders and fetal infection are important etiology of BVM. CMA is highly recommended for genetic disorders’ evaluation. Pregnancies with severe BVM always ended in TOP, while in mild-to-moderate NIBVM, prenatal imaging by ultrasound and/or MRI plays important roles in the pregnancy outcomes.

Isolated Increased Nuchal Translucency in First Trimester Ultrasound Scan: Diagnostic Yield of Prenatal Microarray and Outcome of Pregnancy.

Background: Increased nuchal translucency (NT) is associated with aneuploidy. When the karyotype is normal, fetuses are still at risk for structural anomalies and genetic syndromes. Our study researched the diagnostic yield of prenatal microarray in a cohort of fetuses with isolated increased NT (defined as NT ≥ 3.5 mm) and questioned whether prenatal microarray is a useful tool in determining the adverse outcomes of the pregnancy.Materials and Methods: A prospective study was performed, in which 166 women, pregnant with a fetus with isolated increased NT (ranging from 3.5 to 14.3 mm with a mean of 5.4 mm) were offered karyotyping and subsequent prenatal microarray when karyotype was normal. Additionally, all ongoing pregnancies of fetuses with normal karyotype were followed up with regard to postnatal outcome. The follow-up time after birth was maximally 4 years.Results: Totally, 149 of 166 women opted for prenatal testing. Seventy-seven fetuses showed normal karyotype (52%). Totally, 73 of 77 fetuses with normal karyotype did not show additional anomalies on an early first trimester ultrasound. Totally, 40 of 73 fetuses received prenatal microarray of whom 3 fetuses had an abnormal microarray result: two pathogenic findings (2/40) and one incidental carrier finding. In 73 fetuses with an isolated increased NT, 21 pregnancies showed abnormal postnatal outcome (21/73, 28.8%), 29 had a normal outcome (29/73, 40%), and 23 were lost to follow-up (23/73, 31.5%). Seven out of 73 live-born children showed an adverse outcome (9.6%).Conclusions: Prenatal microarray in fetuses with isolated increased NT had a 5% (2/40) increased diagnostic yield compared to conventional karyotyping. Even with a normal microarray, fetuses with an isolated increased NT had a 28.8% risk of either pregnancy loss or an affected child.

Immature Granulocyte Percentage in Early Chronic Myeloid Leukemia

A 38-year-old man presented with mild fatigue of recent onset to the medical outpatient department (OPD). Clinical examination showed an absence of fever, pallor, and no organomegaly. Routine complete blood count (CBC) showed mild leukocytosis (total leukocyte count 22.93 × 109/L), absence of features of sepsis but with immature granulocyte (IG) fraction of 26.6% on peripheral blood film without basophilia or eosinophilia. A diagnosis of chronic myeloid leukemia (CML) was suspected which was confirmed by conventional karyotyping and quantitative reverse transcription − polymerase chain reaction (QPCR). Following administration of imatinib for 2 weeks, a follow-up CBC showed a reduction in IG fraction to 1.5%. The patient is under follow-up in OPD for 4 months and is responding well as substantiated by follow-up QPCR. To the best of our knowledge, this is the first ever report where CML was first suspected on the basis of IG fraction and the same parameter proved useful for follow-up.

Prenatal diagnosis of Pallister-Killian syndrome and literature review.

Pallister‐Killian syndrome (PKS) is a rare sporadic genetic disorder usually caused by mosaicism of an extra isochromosome of 12p (i(12p)). This retrospective study analysed the prenatal ultrasound manifestations and molecular and cytogenetic results of five PKS foetuses. Samples of amniotic fluid and/or cord blood, skin biopsy and placenta were collected. Conventional karyotyping and single nucleotide polymorphism array (SNP array) were performed on all the amniotic fluid or cord blood samples. Copy number variants sequencing (CNV‐seq) and fluorescence in situ hybridization (FISH) were also used for the validation for one foetus. All the five foetuses were from pregnancies with advanced parental age. Two foetuses involved structural abnormalities and one foetus had only soft markers, all of which included increased nuchal translucency. The rest two foetuses had normal ultrasounds in the second trimester, which has rarely been reported before. The karyotype revealed typical i(12p) in four cases and a small supernumerary marker chromosome consisting of 12p and 20p in the remaining one case. The proportion of cells with i(12p) ranged from 0 to 100% in cultural cells, while SNP array results suggested 2−4 copies of 12p. For one foetus, metaphase FISH showed normal results, but the interphase FISH suggested cell lines with two, three and four copies of 12p in the amniotic fluid. Advanced parental age may be an important risk factor for PKS, and there were no typical ultrasound manifestations related to PKS. A combination of karyotype analysis and molecular diagnosis is an effective method for the diagnosis of PKS.

P–558 SNP array versus karyotype for prenatal diagnosis in fetuses with abnormal ultrasound: a systematic review and meta-analysis

Abstract Study question Does Single nucleotide polymorphism (SNP) array provide a diagnostic advantage over conventional karyotype in prenatal diagnosis for fetuses with an abnormal ultrasound? Summary answer SNP array in the prenatal setting provides an incremental diagnostic yield over karyotype and the diagnostic accuracy is comparable with combined SNP array and karyotype What is known already Single nucleotide polymorphism and comparative genomic hybridization based arrays (aCGH) are the two chromosomal microarray (CMA) platforms available. Guidelines which recommend offering CMA instead of karyotyping for prenatal diagnosis are mainly based on studies that compared aCGH with karyotype. There is a paucity of reviews that critically appraise the role of SNP array as a prenatal diagnostic tool. We decided to estimate the incremental yield of SNP array over karyotype in detecting chromosomal abnormalities, and to determine the diagnostic accuracy of SNP alone compared with SNP array and karyotype in combination for prenatal diagnosis in fetuses with an abnormal ultrasound. Study design, size, duration We conducted a systematic review of studies comparing SNP array with karyotype for prenatal diagnosis in fetuses with an abnormal ultrasound. We performed a literature search in the electronic databases of EMBASE, PubMed, CENTRAL, CDSR, SCOPUS and Web of science for relevant studies published in the English language between January1996 and May 2020. We also hand searched the referenced list of included studies and performed a google search for grey literature to identify potential studies. Participants/materials, setting, methods The study population was women undergoing prenatal diagnosis for abnormal fetal ultrasound. Studies in which SNP array and karyotyping had been used in fetuses with abnormal ultrasound and which allowed for a 2 x 2 data extraction table were included. We estimated the incremental yields for SNP array over karyotype. For determining the diagnostic accuracy, we considered SNP array alone as the index test and combined karyotype &amp; SNP array as the reference standard. Main results and the role of chance We included six studies for quantitative analysis. After pooling results, incremental yield of SNP array over normal karyotype was 10% (95% confidence interval, CI 4 to 16%) in fetuses with abnormal ultrasound while incremental yield of karyotype over SNP array was 1% (95% CI 0 to 2%). The agreement between SNP array and karyotype was 92%. Variant of uncertain significance (VUS) rates ranged from 4–8%. For SNP array alone versus combined SNP array and conventional karyotype, pooled sensitivity and specificity was 0.96 (95% CI 0.91 to 0.99) and 1.00 (95% CI 0.99 to 1.00) respectively. The Area under curve (AUC) was 0.99 indicating the discriminating ability of the SNP array was very high to identify the fetus with chromosomal abnormalities. Limitations, reasons for caution Only studies published in English were included. There was some degree of heterogeneity in inclusion criteria for the included studies. Wider implications of the findings: The current study suggests SNP array alone can replace conventional karyotype for prenatal diagnosis in fetus with an abnormal ultrasound. Limitations to adoption of SNP testing might include the requirement of requisite expertise to interpret the results and counsel patients appropriately, especially with the propensity of SNPs to identify VUS. Trial registration number Not applicable

P–540 A feasible diagnostic approach for the cryptic subtelomeric traslocations in early recurrent miscarriage patients by preimplantation genetic testing (PGT)

Abstract Study question Could cryptic subtelomeric traslocations in early recurrent miscarriage patients be diagnosed by preimplantation genetic testing? Summary answer PGT is a powerful tool to detect subtelomeric cryptic traslocations identifying the cause of early recurrent miscarriage and allowing subsequent genetic counselling. What is known already: Chromosome translocations are frequently associated with birth defects, spontaneous early pregnancy losses and infertility. However, submicroscopic traslocations (so-called cryptic traslocations) are too small to be detected by conventional karyotyping.. Due to balanced status, high resolution molecular techniques as arrayCGH are not able to detect it. Thus, cryptic traslocations detection is challenging. PGT is able to detect CNVs at higher resolution than routine karyotyping. Therefore, the recurrent diagnosis of CNV at embryo level could suggest a subchromosomal parental traslocation. The aim of this study is to investigate the feasibility of using PGT as an indicator of parental balanced cryptic traslocations. Study design, size, duration We included three couples who underwent PGT for unexplained repeated pregnancy loss (RPL) in our clinic from February 2020 to November 2020. Common established causes of RPL (uterine anomalies, antiphospholipid syndrome, immunological, hormonal and metabolic disorders) were previously rouled-out. Even couple karyotypes were normal. Twenty-three embryos from those couples were biopsied at blastocyst and analysed for CNVs detection using low coverage whole genome NGS. Participants/materials, setting, methods PGT by NGS was performed by Veriseq-NGS (Illumina), with previous whole genome amplification. Fluorescence in situ hybridization (FISH) using parental blood samples were performed to validate the origin of subchromosomal number variation. Commercially available subtelomeric specific probes were selected according to the CNV identified and the procedures were performed according to the manufacturer’s protocols. Main results and the role of chance Overall, CNVs of terminal duplication and deletion that imply unbalanced traslocation derivatives were detected in the 43.5% of biopsied embryos. For couple 1, 4 out of 5 embryos (80%) carried deletion of telomeric region on chromosomes 5 and 21. Three out of 6 biopsed embyos (50%) were diagnosed with subchromosomal copy variants at telomeric region on chromosomes 6 and 16 for couple 2. In the case of couple 3, three out of 12 embryos (25%) were carriers of CNV at subtelomeric region on chromosomes 2 and 6. The size of CNVs detected ranges from 8Mb to 20Mb. Accurate diagnosis with the parental study was made by FISH. The combination of probes to detect the structural chromosome alteration were: Tel5qter-LSI21q, Tel6pter-CEP16 and Tel6pter-CEP6 for each couple respectively. The FISH studies reveal that CNVs were inherited from one parent carrying the balanced cryptic traslocation. Ultimately, the abnormal karyotype from the carrier parent were 46,XY,t(5;21)(q33.2;q21.2) for couple 1, 46,XY,t(6;16)(p22.3;q22.1) for couple 2 and 46,XY,t(2;6)(p25.1;p24.2) for couple 3. Finally, each couple performed a cryotransfer of a single normal balanced embryo. Two pregnancies are ongoing. Limitations, reasons for caution The main limitation of this approach is the NGS- PGT resolution. CNVs smaller than 5Mb could not be detected. Wider implications of the findings: This study shows the value of PGT for unexplained RPL, followed by parental FISH to better characterize CNVs and identify couples in whom one partner carries a cryptic translocation. Accurate diagnosis of parental chromosome translocation can achieve with FISH only, but FISH would not be performed unless PGT showed CNVs. Trial registration number Not applicable

Diagnostic Utility of Gene Fusion Panel to Detect Gene Fusions in Fresh and Formalin-Fixed, Paraffin-Embedded Cancer Specimens

Somatic gene fusions are common in leukemias/lymphomas and solid tumors. The detection of gene fusions is crucial for diagnosis. NanoString fusion technology is a multiplexed hybridization method that interrogates hundreds of gene fusions in a single reaction. This study's objective was to determine the performance characteristics and diagnostic utility of NanoString fusion assays in a clinical diagnostics laboratory. Validation using 100 positive specimens and 15 negative specimens by a combined reference standard of fluorescence in situ hybridization (FISH)/RT-PCR/next-generation sequencing (NGS) assays achieved 100% sensitivity in leukemias/lymphomas and 95.0% sensitivity and 100% specificity in solid tumors. Subsequently, 214 consecutive clinical cases, including 73 leukemia/lymphoma specimens and 141 formalin-fixed, paraffin-embedded solid tumor specimens, were analyzed by gene fusion panels across 638 unique gene fusion transcripts. A variety of comparator tests, including FISH panels, conventional karyotyping, a DNA-based targeted NGS assay, and custom RT-PCR testing, were performed in parallel. The gene fusion assay detected 31 gene fusions, including 16 in leukemia/lymphoma specimens and 15 in solid tumor specimens. The overall sensitivity, specificity, and accuracy of gene fusions detected by the gene fusion panel in all 329 specimens (validation and consecutive clinical specimens) tested in this study were 94.8%, 100%, and 97.9%, respectively, compared with FISH/RT-PCR/NGS assays. The gene fusion panel is a reliable approach that maximizes molecular detection of fusions among both fresh and formalin-fixed, paraffin-embedded cancer specimens.

The genetic landscape of intellectual disability and epilepsy in adults and the elderly: a systematic genetic work-up of 150 individuals

PurposeGenetic diagnostics of neurodevelopmental disorders with epilepsy (NDDE) are predominantly applied in children, thus limited information is available regarding adults or elderly. MethodsWe investigated 150 adult/elderly individuals with NDDE by conventional karyotyping, FMR1 testing, chromosomal microarray, panel sequencing, and for unresolved cases, also by exome sequencing (nsingle = 71, ntrios = 24). ResultsWe identified (likely) pathogenic variants in 71 cases (47.3%) comprising fragile X syndrome (n = 1), disease-causing copy number (n = 23), and single-nucleotide variants (n = 49). Seven individuals displayed multiple independent genetic diagnoses. The diagnostic yield correlated with the severity of intellectual disability. Individuals with anecdotal evidence of exogenic early-life events (e.g., nuchal cord, complications at delivery) with alleged/unproven association to the disorder had a particularly high yield of 58.3%. Screening for disease-specific comorbidities was indicated in 45.1% and direct treatment consequences arose in 11.8% of diagnosed individuals. ConclusionPanel/exome sequencing displayed the highest yield and should be considered as first-tier diagnostics in NDDE. This high yield and the numerous indications for additional screening or treatment modifications arising from genetic diagnoses indicate a current medical undersupply of genetically undiagnosed adult/elderly individuals with NDDE. Moreover, knowledge of the course of elderly individuals will ultimately help in counseling newly diagnosed individuals with NDDE.

Higher CNV Frequencies in Chromosome 14 of Girls With Turner Syndrome Phenotype.

Precise genotype-phenotype correlations in Turner syndrome (TS) have not yet been deciphered. The chromosomal basis of the clinical TS phenotype in the absence of X chromosome aberrations on conventional karyotyping remains more and less unexplored. To elucidate the high-resolution chromosomal picture and analyze the genotype-phenotype associations in girls with clinical phenotype of TS by chromosomal microarray. Cross sectional observational study conducted between October 2018 and January 2020 on 47 girls presenting the clinical TS phenotype and fulfilling the criteria for chromosomal analysis. Outpatient department at Department of Endocrinology and the Molecular Research Lab at tertiary care teaching institution. The copy number variation (CNV) polymorphs were more frequent on autosomes than X chromosomes, and they were detected in 89.3%, 61.7%, and 92.8% of patients, respectively, on chromosome 14 or X or both. A total 445 and 64 CNV polymorphs were discovered on chromosome X and 14, respectively. The latter exhibited either gain at 14q32.33, loss at 14q11.2, or both. Karyotype was available for 27 patients; 55.6% of cases displayed X chromosome abnormalities while 44.4% cases had a normal karyotype. Functional interactomes of the genes that were present in chromosome 14 CNVs and those known to be associated with TS showed an overlap of 67% and enriched various development-related cellular pathways underlying TS phenotype. On high-resolution karyotype analysis, clinical phenotype of TS can be associated with CNV defects in autosomes, specifically chromosome 14 or X chromosome or both. The syndrome of chromosome 14 CNV defects with and without X-chromosomal defects clinically mimics TS and shares a common genomic network that deserves further investigations.

BTK, NUTM2A, and PRPF19 Are Novel KMT2A Partner Genes in Childhood Acute Leukemia.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with acute leukemias, especially in infants. KMT2A is rearranged with a big variety of partner genes and in multiple breakpoint locations. Detection of all types of KMT2A rearrangements is an essential part of acute leukemia initial diagnostics and follow-up, as it has a strong impact on the patients’ outcome. Due to their high heterogeneity, KMT2A rearrangements are most effectively uncovered by next-generation sequencing (NGS), which, however, requires a thorough prescreening by cytogenetics. Here, we aimed to characterize uncommon KMT2A rearrangements in childhood acute leukemia by conventional karyotyping, FISH, and targeted NGS on both DNA and RNA level with subsequent validation. As a result of this comprehensive approach, three novel KMT2A rearrangements were discovered: ins(X;11)(q26;q13q25)/KMT2A-BTK, t(10;11)(q22;q23.3)/KMT2A-NUTM2A, and inv(11)(q12.2q23.3)/KMT2A-PRPF19. These novel KMT2A-chimeric genes expand our knowledge of the mechanisms of KMT2A-associated leukemogenesis and allow tracing the dynamics of minimal residual disease in the given patients.

Cytogenetic findings of ectopic endometriotic tissue in women with endometriosis and review of the literature

ObjectiveTo determine chromosome and gene alterations in ectopic endometrial (EM) tissue which may be implicated in the clinical course or the progression of endometriosis and to review the literature concerning the cytogenetic findings of women with endometriosis. Study design15 women who underwent laparoscopic endometriosis surgery at the Athens Genesis Clinic were enrolled in the study. Ectopic endometrial tissue was surgically removed and further analyzed by conventional and molecular cytogenetic techniques. Fluoresent in situ hibridization (FISH) with probes for p53, ATM, MYC, MLL1 and IGH genes, the centromeres of chromosomes 7 and 8 and 7q22/7q31 chromosomal regions was carried out. ResultsKaryotypic analysis revealed no clonal chromosomal abnormalities. However, an increased frequency of polyploidy (55.6%) and sporadic chromosomal abnormalities (40.0%) concerning chromosomes 9, 11, 17 and X were noticed involving mainly deletions, trisomies or monosomies. FISH analysis showed IGH gene rearrangements in 54% of the EM cases and MLL gene rearrangements in 73% of the examined samples. Normal hybridization patterns were observed for p53, ATM and MYC. The increased frequency of polyploidy shown by conventional karyotyping was also confirmed by FISH. ConclusionPolyploidy, sporadic chromosomal abnormalities, as well as IGH and MLL gene rearrangements, may provoke genetic instability and play a potential role in the development of endometriosis. IGH and MLL gene rearrangements indicate a genetic relation between endometriosis and carcinogenesis. Confirmation of the above gene rearrangements in a large series of women may allow the determination of their possible involvement in the pathogenesis of this complex disease and their possible contribution in the early identification of women in danger for malignant transformation.

A High- Risk Genetic Signature Is Predictive For Poor Outcome In Auto-Transplant Eigible Multiple Myeloma Patients Even With Use Of Novel Agents. A Single Institution Study

IntroductionMulti agent induction therapy followed by an auto transplant is currently the standard of care in fit patients up to the age of 70 in most haematology units. There is little data available on the actual real world uptake of this “standard of care” as many series have a significant selection bias. We were able to ascertain the true uptake of an auto transplant in a large, continuous series of all the patients referred to a single unit. AimThis study analysed the outcomes of a cohort of auto transplant eligible patients (defined as up to age 68) over a decade of treatment at one institution. Genetic analysis by karyotype (G – banding) and FISH was performed in all patients and high risk abnormalities identified. We then compared survival in the transplant and non transplant groups and performed a subset analysis according to possession of a high risk genetic signature. MethodsThe multiple myeloma database at Wellington Hospital was interrogated during the period 2002 to 2012. One hundred and forty patients were identified with an age range from 32 to 68 years. There was a male preponderance of 89 and 51 females. Conventional chromosome karyotype was done and 13q deletions were detected by this method and considered high risk. The FISH panel was comprehensive and included 13q del., t(4:14), t(14:16), p53 deletion. In the last five years 1Q gains, MYC and a hyper-diploid clone were also routine. ResultsThe 140 patients were divided into 80 receiving an auto graft and 68 did not for various reasons. The reasons included refusal, co-morbid conditions such as cardiac, respiratory and significant renal impairment. Two patients who were on dialysis were auto grafted however and not excluded.Induction treatment over the decade included VAD initially, then cyclophosphamide/ dexamethasone and then from 2008, a bortezomib based regime of Cy/Bor/D was employed. Cy/Bor/D induced patients numbered 40 or 50% of the total auto graft cohort.There were 25% high risk abnormalities detected in the auto graft group and t(4:14) was detected in 8% and p53 del. totalled 6% (6 of 80). The transplant related mortality in this group was 0% (no deaths within 100 days). The overall survival (OS) at three years was 62% and this compared with an OS of 30% in the non transplant group (P = 0.001). The non auto graft group also had a 25% (15 out of 60 ) bad risk signature rate which included three patients with t(4:14), six p53 del. (12%), one t(4:14) case and complex karyotypes in five. One patient had a t(4:14), a p53 deletion and a 1Q gain (triple hit). This patient subgroup had no survivors at the three year mark. The median OS of the non auto graft group was 16 months and has not been reached in the transplant group. ConclusionThis group of auto grafted myeloma patients has a TMR of zero which represents some of the best results reported. They had an excellent three year OS of 62% which is projected to be eventually greater than 75% when the more recently treated patients cross the three year mark.The use of bortezomib in the latest auto grafted cohort currently includes only two deaths, so this drug has resulted in CR rates approaching 40% when assessed by day 100 marrows. This study illustrates real world data and clearly shows that an auto transplant confers a significant survival advantage. The possession of a bad risk signature however is the most important predictor of an adverse outcome whether or not patients receive a transplant. Disclosures:No relevant conflicts of interest to declare.

Conventional Karyotyping Is Still an Important Prognostic Marker for Myeloma in the Era of R-ISS Staging

Introduction: The Revised International Staging System (R-ISS) has been widely adopted to prognosticate Multiple Myeloma (MM). As a result, the continued utility of conventional karyotyping, has been called into question.Methods: A multi-centre study for newly diagnosed MM patients who received novel agent/s at induction from year 1999 at the National University Hospital Singapore, Singapore General Hospital, Alfred Hospital, Melbourne, Australia, was conducted. Conventional karyotype information was categorized based on ploidy as follows: normal, hyperdiploid (48-74 chromosome numbers), non-hyperdiploid (other abnormal karyotype). We evaluated the impact of ploidy on Overall Survival (OS) including a multivariate analysis, taking into account the R-ISS stages, transplant status, age, and novel agent/s used at induction. We also evaluated if it is possible to substitute FISH with conventional karyotyping in identifying high-risk patients. Results were validated in an independent cohort from China.Results: There were 303 patients evaluable. Ploidy significantly affected OS of patients with R-ISS stage II (N=212; p=0.026), with non-hyperdiploid patients doing worst. For R-ISS stage I patients (N=20), the number of patients in each cytogenetic group was too small to draw any conclusion. Non-hyperdiploid karyotype had no prognostic impact on patients with R-ISS stage III (N=71; p=0.990). In the multivariate analysis, ploidy was significantly associated with OS (p=0.018).Based on our results, we divided the R-ISS stage II patients into groups with or without non-hyperdiploid karyotype. The R-ISS stage I group and the R-ISS stage II group without non-hyperdiploid karyotype had similar median OS (median OS of 92.1 and 96.0 months respectively; p=0.001). They were grouped together into a standard-risk group. The R-ISS stage II group with non-hyperdiploid karyotype and the R-ISS stage III group had similar OS and were categorized together as a high-risk group (median OS of 45.0 months and 45.1 months respectively; p=0.001). As high-risk myeloma are of most clinical significance, we proposed a new prognostic staging system that incorporates ploidy into the R-ISS. In this R-ISS+K(Karyotype) staging system, patients were divided into standard and high-risk groups.. Patients in the 2 groups had significantly different OS (median OS of 92.1 versus 45.0 months, p=0.000).We replaced FISH with conventional karyotyping in the K(Karyotype)-ISS staging system, where stage I consisted of patients with ISS stage I with normal LDH and cytogenetics, stage III consisted of patients with ISS stage III with raised LDH or abnormal cytogenetics, and stage II consisted of all others. Of the R-ISS stage III patients, 83.0% were also K-ISS stage III. R-ISS stage II patients who were K-ISS Stage III had median OS of 45.0 months, shorter than K-ISS stage II patients that were R-ISS stage III (median OS of 60.0 months). In this K-ISS staging system, K-ISS stage III patients had median OS of 38.0 months, shorter than the median OS (45.1 months) of the R-ISS stage III group.These results were validated with an independent cohort of 382 patients from the Institute of Hematology, Peking University, China. The median OS in the R-ISS+K standard risk group was 127.1 months compared to 43.0 months for the high risk group (p=0.000). Similarly, K-ISS stage III patients had median OS of 43.1 months, compared to 46.1 months for the R-ISS stage III patients (p=0.000).Conclusion: Ploidy by conventional karyotyping is an independent prognostic factor even in the setting of R-ISS. Ploidy can be incorporated into R-ISS staging system to better identify high-risk patients (R-ISS+K). In countries where FISH is not available, conventional cytogenetics can be used instead of FISH (K-ISS) to identify high-risk patients. [Display omitted] DisclosuresSpencer:Amgen: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chng:Janssen China R&D: Research Funding.

The Gain of the Short Arm of Chromosome 2 (2pgain) Plays an Important Role in CLL Drug Resistance, and Has to be Systematically Considered before Deciding an Adapted Therapy

The Gain of the Short Arm of Chromosome 2 (2pgain) Plays an Important Role in CLL Drug Resistance, and Has to be Systematically Considered before Deciding an Adapted Therapy

Cytogenetics-Based Risk Prediction of Blastic Transformation of CML Treated with Tyrosine Kinase Inhibitors

Background:Blast phase (BP) of chronic myeloid leukemia (CML) remains a major therapeutic challenge in the era of tyrosine kinase inhibitor (TKI) therapy. Given the lack of effective treatment for BP, a potential key to improve patient outcome lies in identifying patients at high risk of progression and preventing onset of BP. Additional chromosomal abnormalities (ACAs) are important determinants of patient outcome. Despite its rarity in TKI era, BP develops in most CML patients in pre-TKI era regardless of ACAs. It is unknown how the development of BP is differently affected by ACAs in the TKI era.Design:We investigated the value of ACAs in prediction of BP in a large cohort of CML patients. Inclusion criteria included: 1, received TKIs as part of therapy; 2, age at diagnosis > 18 years; 3, presence of t(9;22) or variant translocations detected by conventional karyotyping. All cytogenetic abnormalities in Ph+ cells except -Y were considered ACAs. We examined the latencies from initial CML diagnosis to ACA emergence (Interval 1), from ACA emergence to BP (Interval 2) and survival after BP (Interval 3).Results:A total of 2,326 patients were included in the study, including 1,322 men and 1,004 women with a median age at diagnosis of 49 years (range, 18-88 years). In total, 570 had ACAs, including 333 (58.4%) with a single ACA and 237 (41.5%) with multiple ACAs. The most common single ACAs were: +8, +Ph, 3q26.2 rearrangement, i(17q), and -7/7q-, with a frequency of 13.5%, 12.6%, 7.5%, 6.6%, 3.9%, respectively. Of 237 patients with complex ACAs, 18 had 3q26.2 rearrangement, 19 had -7/7q-, and 30 had i(17q) as a component of complex ACAs. Seven patients had two of these three ACAs in different combinations. These complex ACAs were grouped together as high-risk complex ACAs. Complex ACAs without these three chromosomal abnormalities were designated as other complex ACAs.Patients with 3q26.2 rearrangement, -7/7q-, i(17q) and high-risk complex ACAs had a significantly faster blastic transformation, with a median Interval 2 of 2.8, 0, 32.6, 0 months, respectively, compared with 19.2 months in patients with other complex ACAs, and unreached in patients with +8, +Ph, or other single ACAs, and (Fig 1A). Based on the frequency and velocity of blastic transformation, patients were stratified into four risk groups. Patients without ACA formed the standard-risk (SR) group, patients with isolated 3q26.2 rearrangement, -7/7q-, or i(17q) and high-risk complex ACAs formed the high-risk (HR) group, patients with +8, +Ph or other single ACAs formed the intermediate-1 risk (IR-1) group, and patients with other complex ACAs formed the intermediate-2 risk (IR-2) group. By risk group, the median Interval 2 was unreached for IR-1group, 19.2 months for IR-2 group, and 1.9 months for HR group. (Fig 1B), and the interval from CML diagnosis to BP (Interval 1 + Interval 2) was unreached for SR and IR-1, 83.7 months for IR-2 patients, and 58.2 months for HR patients (Fig 1C). This risk of BP correlated well with patient survival from CML diagnosis (Fig 1D).The disease course of CML patients with ACAs follows a triphasic course, that is, Interval 1, 2 and 3. There was no or little variation in Interval 1 among three ACA groups (Fig 1E). No significant difference in Interval 3 was observed among three ACA groups neither (Fig 1F). SR patients had only slightly better survival of 17.0 months, compared with 9.3 months in other 3 groups combined (Fig 1F). The lack of difference in Interval 1 and 3 indicate that the predominant determinant of CML disease course in TKI era lies in the duration of Interval 2, which is in turn highly ACA-dependent.Conclusions:Chromosomal alterations predict the risk of blastic transformation in CML patients treated with TKIs. Patients can be stratified into four different cytogenetic risk groups based on ACA-dependent risk of blastic transformation. This risk stratification highly correlates with patient survival. By prolonging the duration of Interval 2, TKI therapy mitigates the risk of blastic transformation associated with low-risk ACAs or no ACAs but does not alter the natural course of CML associated with high-risk ACAs: 3q26.2 rearrangement, -7/7q- and i(17q). Thus, we identify a group of patients who are at a high risk of rapid disease progression. These patients may benefit from alternative treatment approach including early allogeneic hematopoietic stem cell transplantation to prevent blastic transformation. [Display omitted] DisclosuresCortes:ARIAD: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Teva: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Sun Pharma: Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding. Kantarjian:Bristol-Meyers Squibb: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Delta-Fly Pharma: Research Funding.