Molecular Karyotyping Research Articles

Benign, Pathogenic and Copy Number Variations of Unknown Clinical Significance in Patients with Congenital Malformations and Developmental Delay.

The high frequency (3.0-5.0%) of congenital anomalies (CA) and intellectual disabilities (IDs), make them a serious problem, responsible for a high percentage (33.0%) of neonatal mortality. The genetic cause remains unclear in 40.0% of cases. Recently, molecular karyotyping has become the most powerful method for detection of pathogenic imbalances in patients with multiple CAs and IDs. This method is with high resolution and gives us the opportunity to investigate and identify candidate genes that could explain the genotype-phenotype correlations. This article describes the results from analysis of 81 patients with congenital malformations (CMs), developmental delay (DD) and ID, in which we utilized the CytoChip ISCA oligo microarray, 4 × 44 k, covering the whole genome with a resolution of 70 kb. In the selected group of patients with CAs, 280 copy number variations (CNVs) have been proven, 41 were pathogenic, 118 benign and 121 of unknown clinical significance (average number of variations 3.5). In six patients with established pathogenic variations, our data revealed eight pathogenic aberrations associated with the corresponding phenotype. The interpretation of the other CNVs was made on the basis of their frequency in the investigated group, the size of the variation, content of genes in the region and the type of the CNVs (deletion or duplication).

Array-based molecular karyotyping in 115 VATER/VACTERL and VATER/VACTERL-like patients identifies disease-causing copy number variations.

The acronym VATER/VACTERL refers to the rare nonrandom association of the following component features (CF): vertebral defects (V), anorectal malformations (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia, renal malformations (R), and limb defects (L). Patients presenting with at least three CFs are diagnosed as having VATER/VACTERL association while patients presenting with only two CFs are diagnosed as having VATER/VACTERL-like phenotypes. Recently, rare causative copy number variations (CNVs) have been identified in patients with VATER/VACTERL association and VATER/VACTERL-like phenotypes. To detect further causative CNVs we performed array based molecular karyotyping in 75 VATER/VACTERL and 40 VATER/VACTERL-like patients. Following the application of stringent filter criteria, we identified 13 microdeletions and seven microduplications in 20 unrelated patients all of which were absent in 1,307 healthy inhouse controls (n < 0.0008). Among these, microdeletion at 17q12 was confirmed to be de novo. Three microdeletions at 5q23.1, 16q23.3, 22q11.21, and one microduplication at 10q11.21 were all absent in the available parent. Microdeletion of chromosomal region 22q11.21 was previously found in VATER/VACTERL patients rendering it to be causative in our patient. The remaining 15 CNVs were inherited from a healthy parent. In two of 115 patients' causative CNVs were found (2%). The remaining identified rare CNVs represent candidates for further evaluation. Rare inherited CNVs may constitute modifiers of, or contributors to, multifactorial VATER/VACTERL or VATER/VACTERL-like phenotypes. Birth Defects Research 109:1063-1069, 2017. © 2017 Wiley Periodicals, Inc.

High molecular karyotype variation revealed in indigenous Elymus nutans in the Qinghai Plateau

The karyotypes of 27 individuals of Elymus nutans from eight wild populations in the Qinghai Plateau were analyzed using sequential FISH and GISH. High FISH pattern polymorphism and karyotype variation were detected within and among populations. The chromosome variations were mainly characterized as repeat deletions and amplifications along with inter-genomic translocations. The chromosomes of the St and Y genomes demonstrated higher polymorphism than those of the H genome. Six different inter-genomic translocations were identified in 33.3% of individuals; type I and II translocations were detected with higher frequency. Further analysis revealed that type I and II translocations were distributed in different geographic regions. The origin of high karyotype variation of E. nutans in the Qinghai plateau is further discussed.

Novel genes involved in severe early-onset obesity revealed by rare copy number and sequence variants.

Obesity is a multifactorial disorder with high heritability (50–75%), which is probably higher in early-onset and severe cases. Although rare monogenic forms and several genes and regions of susceptibility, including copy number variants (CNVs), have been described, the genetic causes underlying the disease still remain largely unknown. We searched for rare CNVs (>100kb in size, altering genes and present in <1/2000 population controls) in 157 Spanish children with non-syndromic early-onset obesity (EOO: body mass index >3 standard deviations above the mean at <3 years of age) using SNP array molecular karyotypes. We then performed case control studies (480 EOO cases/480 non-obese controls) with the validated CNVs and rare sequence variants (RSVs) detected by targeted resequencing of selected CNV genes (n = 14), and also studied the inheritance patterns in available first-degree relatives. A higher burden of gain-type CNVs was detected in EOO cases versus controls (OR = 1.71, p-value = 0.0358). In addition to a gain of the NPY gene in a familial case with EOO and attention deficit hyperactivity disorder, likely pathogenic CNVs included gains of glutamate receptors (GRIK1, GRM7) and the X-linked gastrin-peptide receptor (GRPR), all inherited from obese parents. Putatively functional RSVs absent in controls were also identified in EOO cases at NPY, GRIK1 and GRPR. A patient with a heterozygous deletion disrupting two contiguous and related genes, SLCO4C1 and SLCO6A1, also had a missense RSV at SLCO4C1 on the other allele, suggestive of a recessive model. The genes identified showed a clear enrichment of shared co-expression partners with known genes strongly related to obesity, reinforcing their role in the pathophysiology of the disease. Our data reveal a higher burden of rare CNVs and RSVs in several related genes in patients with EOO compared to controls, and implicate NPY, GRPR, two glutamate receptors and SLCO4C1 in highly penetrant forms of familial obesity.

Polymorphism of lactose genes in the dairy yeasts Kluyveromyces marxianus, potential probiotic microorganisms

Molecular karyotyping and Southern blot hybridization were used to investigate chromosomal polymorphism of the LAC genes controlling lactose fermentation in Kluyveromyces marxianus strains isolated from various dairy products and natural sources in Russia and CIS countries. Profound polymorphism of karyotype patterns and accumulation of LAC genes were observed in dairy K. marxianus strains. K. marxianus strains isolated from dairy products intensively fermented lactose at 37°C after one day of cultivation, while non-dairy strains exhibited delayed lactose fermentation or did not ferment it at all. Based on the fermentation tests, twelve K. marxianus strains were selected, which are of interest as potential probiotic microorganisms suitable for further molecular genetic studies and breeding.

Cytogenetic and molecular genetic analysis of small supernumerary marker chromosomes in fetal amniotic fluid

To explore the origin and mechanism of small supernumerary marker chromosomes (sSMC) in order to facilitate genetic counseling. Chromosome karyotypes of two fetuses and their immediate family members were analyzed by conventional G banding. High-throughput whole genome sequencing was used to determine the origin of sSMCs. Fetus 1 was shown to have a karyotype of 47,XY,+mar but with normal FISH and B ultrasound findings. Its father also had a 47,XY,+mar karyotype with normal FISH results and clinical phenotype. High-throughput genome sequencing revealed that fetus 1 and its father were both 46,XY,dup(21)(q11.2;q21.1) with a 6.2 Mb duplication of the long arm of chromosome 21. The fetus was born with normal phenotype and developed well. Its grandmother also had a karyotype of 46,XX,t(15;21)(q13;p13) with normal FISH result and clinical phenotype. The karyotypes of its mother and grandfather were both normal. Analysis of fetus 2 showed a 47,XY,+mar karyotype with normal FISH results. High-throughput genome sequencing suggested a molecular karyotype of 46,XX. The fetus was born with normal phenotype and developed well. The karyotypes of its parents were both normal. Considering their variable origins, identification of sSMC should combine conventional G banding analyses with high-throughput whole genome sequencing for precise delineation of the chromosomes.

Fibrous hamartoma of infancy: a clinicopathologic study of 145 cases, including 2 with sarcomatous features

Fibrous hamartoma of infancy is a rare soft tissue lesion of infants and young children with characteristic triphasic morphology, which typically occurs in the axilla and less commonly in other locations. We reviewed 145 cases of fibrous hamartoma of infancy from our consultation archives. Cases occurred in 106 males and 39 females (mean age—15 months; range—birth to 14 years), and involved both typical sites (eg, axilla/back/upper arm) (n=69) and unusual locations (n=76). Six were congenital. The tumors presented as subcutaneous masses and ranged from 0.4 to 17 cm (mean 3 cm). All displayed triphasic morphology, but varied widely in the relative percentages of fat, fibroblastic fascicles, and primitive mesenchyme. Hyalinized zones with cracking artifact, mimicking giant cell fibroblastoma, were present in a 44 (30%) of cases; however FISH for PDGFB gene rearrangement was negative in five tested cases. In addition to classical fibrous hamartoma of infancy, two lesions contained large sarcomatous-appearing foci with high cellularity, high nuclear grade, and brisk mitotic activity. One occurred in a 10-month-old female as a new mass in a congenital fibrous hamartoma of infancy; the other occurred as a leg mass in a 6-year-old male. ETV6 gene rearrangement was negative in the tumor from the 10-month-old female. Genomic microarray (OncoScan) showed normal molecular karyotype in eight tested cases, whereas the two tumors with sarcomatous features showed a hyperdiploid/near tetraploid molecular karyotype with copy neutral loss of heterozygosity of chromosomes 1p and 11p, and loss of 10p, chromosome 14, and a large portion of chromosome 22q (22q11.23q13.33), respectively. Follow-up (52 patients; range: 1–208 months, median: 8 months) showed only two local recurrences and no metastases. Extensive local disease in the 10-month-old female with sarcomatous-appearing fibrous hamartoma of infancy necessitated forequarter amputation. In summary, our study confirms the classic clinicopathologic features, including the triphasic morphologic appearance of most cases. In contrast to earlier studies, our series illustrates a broader histologic spectrum than previously appreciated, including its close resemblance to giant cell fibroblastoma in one quarter of cases and the rare presence of ‘sarcomatous' areas, the latter providing evidence that these are complex neoplasms rather than hamartomas.

Repetitive DNA: A Versatile Tool for Karyotyping in Festuca pratensis Huds.

FISH is a useful method to identify individual chromosomes in a karyotype and to discover their structural changes accompanying genome evolution and speciation. DNA probes for FISH should be chromosome specific and/or exhibit specific patterns of distribution along each chromosome. Such probes are not available in many plants including meadow fescue (Festuca pratensis Huds.), an important forage grass species. In the present study, various DNA repeats identified in Illumina shotgun sequences specific to chromosome 4F of F. pratensis were used as probes for FISH to develop the molecular karyotype of meadow fescue and to reveal a long-range molecular organization of its chromosomes. Five tandem repeats produced specific patterns on individual chromosomes. Their use in combination with probes for rRNA genes enabled the establishment of the molecular karyotype of meadow fescue. Most of the mobile genetic elements were dispersed along all the chromosomes except for the DNA transposon CACTA, which was localized preferentially to telomeric and subtelomeric regions, and a putative LTR element, which was localized to (peri)centromeric regions. Cytogenetic mapping of the 5 tandem repeats in other accessions of meadow fescue showed a highly similar distribution and confirmed the versatility and robustness of these probes.

Chorionic villus sampling in the cell‐free DNA aneuploidy screening era: careful selection criteria can maximise the clinical utility of screening and invasive testing

To quantify the impact of cell-free DNA (cfDNA) screening on chorionic villus sampling (CVS) test indications and outcomes in a tertiary maternity service. Retrospective cohort study of all CVS procedures performed for any indication on singleton pregnancies at The Royal Women's Hospital, Melbourne, and at Women's Ultrasound Melbourne, Australia, between August 2008 and February 2015. Karyotypes were classified according to pathogenicity and detectability by standard cfDNA screening panels. A total of 2051 CVS procedures, 25 373 twelve-week scans and 2394 cfDNA tests were performed. The CVS rate per 12-week scan fell from 9.8 to 3.9% following introduction of cfDNA screening. The yield of pathogenic chromosomal anomalies per CVS increased from 12.9 to 25.2%, with 70% of pathogenic results now comprising T21, up from 52%. Sixteen (5.3%) of the pathogenic chromosomal abnormalities identified on CVS would not have been predicted by current cfDNA tests. There is an evolving tension between improved screening performance for common aneuploidies offered by cfDNA testing, and the increasing diagnostic utility of molecular karyotyping. However, the risk of not identifying pathogenic chromosomal abnormalities is low if cfDNA screening is offered in the absence of a structural fetal anomaly, increased nuchal translucency or relevant family history. © 2017 John Wiley & Sons, Ltd.

Comparison of cytogenetics and molecular karyotyping for chromosome testing of miscarriage specimens

To compare chromosome testing of miscarriage specimens between traditional cytogenetic analysis and molecular karyotyping using single nucleotide polymorphism microarrays (SNP) and array comparative genomic hybridization (aCGH). Prospective blinded cohort study. University-based practice. Women undergoing dilation and curettage for first-trimester miscarriage between March 2014 and December2015. None. Chromosome analysis from chorionic villi separated equally and submitted for cytogenetics, SNP microarray, and aCGH testing. Sixty samples were analyzed, of which 47 (78%) were chromosomally abnormal. A correct call was defined when a result was concordant with at least one other testing platform. The correct call rate was 85%, 93%, and 85% using cytogenetics, SNP array, and aCGH, respectively. We found a 33% overall discordance rate between results. Discordances were due to maternal cell contamination, balanced chromosome rearrangements, polyploidy, and placental mosaicism. Mosaicism was detected in 18% of all samples. Growth failure occurred in four samples sent to cytogenetics, of which three were chromosomally abnormal by molecular testing. This study demonstrates the many technical limitations of the three testing modalities. Our rates of maternal cell contamination were low, but it is important to note that this is a commonly reported limitation of cytogenetics. Given the similar overall performance of the three testing modalities, providers may choose a method based on individual availability and consideration of limitations as it applies to each clinical scenario. The unexpected high rate of placental mosaicism warrants further investigation.

Mitochondrial encephalomyopathy and retinoblastoma explained by compound heterozygosity of SUCLA2 point mutation and 13q14 deletion.

Mutations in SUCLA2, encoding the s-subunit of succinyl-CoA synthetase of Krebs cycle, are one cause of mitochondrial DNA depletion syndrome. Patients have been reported to have severe progressive childhood-onset encephalomyopathy, and methylmalonic aciduria, often leading to death in childhood. We studied two families, with children manifesting with slowly progressive mitochondrial encephalomyopathy, hearing impairment and transient methylmalonic aciduria, without mtDNA depletion. The other family also showed dominant inheritance of bilateral retinoblastoma, which coexisted with mitochondrial encephalomyopathy in one patient. We found a variant in SUCLA2 leading to Asp333Gly change, homozygous in one patient and compound heterozygous in one. The latter patient also carried a deletion of 13q14 of the other allele, discovered with molecular karyotyping. The deletion spanned both SUCLA2 and RB1 gene regions, leading to manifestation of both mitochondrial disease and retinoblastoma. We made a homology model for human succinyl-CoA synthetase and used it for structure–function analysis of all reported pathogenic mutations in SUCLA2. On the basis of our model, all previously described mutations were predicted to result in decreased amounts of incorrectly assembled protein or disruption of ADP phosphorylation, explaining the severe early lethal manifestations. However, the Asp333Gly change was predicted to reduce the activity of the otherwise functional enzyme. On the basis of our findings, SUCLA2 mutations should be analyzed in patients with slowly progressive encephalomyopathy, even in the absence of methylmalonic aciduria or mitochondrial DNA depletion. In addition, an encephalomyopathy in a patient with retinoblastoma suggests mutations affecting SUCLA2.

On the allopolyploid origin and genome structure of the closely related species Hordeum secalinum and Hordeum capense inferred by molecular karyotyping.

To provide additional information to the many phylogenetic analyses conducted within Hordeum , here the origin and interspecific affinities of the allotetraploids Hordeum secalinum and Hordeum capense were analysed by molecular karyotyping. Karyotypes were determined using genomic in situ hybridization (GISH) to distinguish the sub-genomes and , plus fluorescence in situ hybridization (FISH)/non-denaturing (ND)-FISH to determine the distribution of ten tandem repetitive DNA sequences and thus provide chromosome markers. Each chromosome pair in the six accessions analysed was identified, allowing the establishment of homologous and putative homeologous relationships. The low-level polymorphism observed among the H. secalinum accessions contrasted with the divergence recorded for the sub-genome of the H. capense accessions. Although accession H335 carries an intergenomic translocation, its chromosome structure was indistinguishable from that of H. secalinum . Hordeum secalinum and H. capense accession H335 share a hybrid origin involving Hordeum marinum subsp. gussoneanum as the genome donor and an unidentified genome progenitor. Hordeum capense accession BCC2062 either diverged, with remodelling of the sub-genome, or its genome was donated by a now extinct ancestor. A scheme of probable evolution shows the intricate pattern of relationships among the Hordeum species carrying the genome (including all H. marinum taxa and the hexaploid Hordeum brachyantherum ).

Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use

BackgroundIncreasing evidence suggests the safety and efficacy of mesenchymal stromal cells (MSC) as advanced therapy medicinal products because of their immunomodulatory properties and supportive role in hematopoiesis. Although bone marrow remains the most common source for obtaining off-the-shelf MSC, cord blood (CB) represents an alternative source, which can be collected noninvasively and without major ethical concerns. However, the low estimated frequency and inconsistency of successful isolation represent open challenges for the use of CB-derived MSC in clinical trials. This study explores whether CB may represent a suitable source of MSC for clinical use and analyzes several in vitro parameters useful to better define the quality of CB-derived MSC prior to clinical application.MethodsCB units (n = 50) selected according to quality criteria (CB volume ≥ 20 ml, time from collection ≤ 24 h) were cultured using a standardized procedure for CB-MSC generation. MSC were analyzed for their growth potential and secondary colony-forming capacity. Immunophenotype and multilineage differentiation potential of culture-expanded CB-MSC were assessed to verify MSC identity. The immunomodulatory activity at resting conditions and after inflammatory priming (IFN-γ-1b and TNF-α for 48 hours) was explored to assess the in vitro potency of CB-MSC prior to clinical application. Molecular karyotyping was used to assess the genetic stability after prolonged MSC expansion.ResultsWe were able to isolate MSC colonies from 44% of the processed units. Our results do not support a role of CB volume in determining the outcome of the cultures, in terms of both isolation and proliferative capacity of CB-MSC. Particularly, we have confirmed the existence of two different CB-MSC populations named short- and long-living (SL- and LL-) CBMSC, clearly diverging in their growth capacity and secondary colony-forming efficiency. Only LL-CBMSC were able to expand consistently and to survive for longer periods in vitro, while preserving genetic stability. Therefore, they may represent interesting candidates for therapeutic applications. We have also observed that LL-CBMSC were not equally immunosuppressive, particularly after inflammatory priming and despite upregulating priming-inducible markers.ConclusionsThis work supports the use of CB as a potential MSC source for clinical applications, remaining more readily available compared to conventional sources. We have provided evidence that not all LL-CBMSC are equally immunosuppressive in an inflammatory environment, suggesting the need to include the assessment of potency among the release criteria for each CB-MSC batch intended for clinical use, at least for the treatment of immune disorders as GvHD.

Detectable clonal mosaicism in blood as a biomarker of cancer risk in Fanconi anemia

AbstractDetectable clonal mosaicism for large chromosomal events has been associated with aging and an increased risk of hematological and some solid cancers. We hypothesized that genetic cancer predisposition disorders, such as Fanconi anemia (FA), could manifest a high rate of chromosomal mosaic events (CMEs) in peripheral blood, which could be used as early biomarkers of cancer risk. We studied the prevalence of CMEs by single-nucleotide polymorphism (SNP) array in 130 FA patients' blood DNA and their impact on cancer risk. We detected 51 CMEs (4.4-159 Mb in size) in 16 out of 130 patients (12.3%), of which 9 had multiple CMEs. The most frequent events were gains at 3q (n = 6) and 1q (n = 5), both previously associated with leukemia, as well as rearrangements with breakpoint clustering within the major histocompatibility complex locus (P = 7.3 × 10−9). Compared with 15 743 age-matched population controls, FA patients had a 126 to 140 times higher risk of detectable CMEs in blood (P < 2.2 × 10−16). Prevalent and incident hematologic and solid cancers were more common in CME carriers (odds ratio [OR] = 11.6, 95% confidence interval [CI] = 3.4-39.3, P = 2.8 × 10−5), leading to poorer prognosis. The age-adjusted hazard risk (HR) of having cancer was almost 5 times higher in FA individuals with CMEs than in those without CMEs. Regarding survival, the HR of dying was 4 times higher in FA individuals having CMEs (HR = 4.0, 95% CI = 2.0-7.9, P = 5.7 × 10−5). Therefore, our data suggest that molecular karyotyping with SNP arrays in easy-to-obtain blood samples could be used for better monitoring of bone marrow clonal events, cancer risk, and overall survival of FA patients.

Genetic stability of human endometrial mesenchymal stem cells assessed with morphological and molecular karyotyping

The aim of this study was to monitor the genetic stability of endometrial mesenchymal stem cells (eMSCs) by G-banding and molecular karyotyping. We evaluated the sensitivity of each method to assess the genetic stability of eMSCs. G-banding karyotyping performed on passages 6 and 15 showed that more than 80% cells had normal karyotype. Random karyotypic changes were found in a small part of the cell population: aneuploidy, isochromosomes, chromosome breakages, interchromosomal association. Molecular karyotyping carried out on the 6th and 14th passages revealed genomic stability, except for in the case of chromosomes 7 and 14. Microduplications 7q36.3 (62 kb) and 14q11.2 (165kb) were found in these chromosomes. We interpreted these aberrations as being derived from the donor of these cells. The morphological and molecular karyotyping complemented each other. Using these methods, we can analyze karyotypic stability at different levels of the genomic organization.

Molecular Genetic Analysis of Human Endometrial Mesenchymal Stem Cells That Survived Sublethal Heat Shock.

High temperature is a critical environmental and personal factor. Although heat shock is a well-studied biological phenomenon, hyperthermia response of stem cells is poorly understood. Previously, we demonstrated that sublethal heat shock induced premature senescence in human endometrial mesenchymal stem cells (eMSC). This study aimed to investigate the fate of eMSC-survived sublethal heat shock (SHS) with special emphasis on their genetic stability and possible malignant transformation using methods of classic and molecular karyotyping, next-generation sequencing, and transcriptome functional analysis. G-banding revealed random chromosome breakages and aneuploidy in the SHS-treated eMSC. Molecular karyotyping found no genomic imbalance in these cells. Gene module and protein interaction network analysis of mRNA sequencing data showed that compared to untreated cells, SHS-survived progeny revealed some difference in gene expression. However, no hallmarks of cancer were found. Our data identified downregulation of oncogenic signaling, upregulation of tumor-suppressing and prosenescence signaling, induction of mismatch, and excision DNA repair. The common feature of heated eMSC is the silence of MYC, AKT1/PKB oncogenes, and hTERT telomerase. Overall, our data indicate that despite genetic instability, SHS-survived eMSC do not undergo transformation. After long-term cultivation, these cells like their unheated counterparts enter replicative senescence and die.

A Rare Familial Paracentric Inversion in the Long Arm of Chromosome 8: Case Report and Review of the Literature

Background/Aim: The incidence of paracentric inversions (PAI)in the general population ranges from 0.09-0.49/1,000 and PAIs involving a relatively short chromosome segment, are generally considered to be harmless. However, PAI carriers have a very low risk to give rise to a viable zygote (3.8%).To present a rare case of a healthy individual, carrier of an 8q paracentric inversion. Methods: A 37-year-old man and his 30-year-old wife were referred for karyotypic analysis due to a previous miscarriage at 33 weeksof gestation. Cytogenetic analysis was performed on peripheralblood lymphocytes by GTG banding. Molecular karyotype analysis performed using Agilent G3 4x180 K CGH+SNP microarray platform. Results: The wife had a normal karyotype 46,XX but the man’s karyotype was 46,XY,inv(8)(q23.1q24.2). Karyotypic analysis ofhis parents revealed that his mother carried the same inversion.Molecular analysis reported no duplication or loss in or near the inverted region. Conclusions: Review of the literature revealed the rarity ofchromosome 8 paracentric inversions and showed that although the risk of a carrier to have an abnormal offspring is low, in cases of large inversions or previous abortions prenatal diagnosis should be offered by conventional and molecular cytogenetic techniques.

A Comprehensive Panel of Molecular Mutations Notably Improves a Cytogenetic Prognostication System in Routine AML Diagnostics

Background: Based on sequencing studies the molecular landscape of AML has been unraveled. Novel prognostic scores combining molecular mutations and karyotype have been proposed (Grimwade et al. Blood, 2016, Döhner et al. NEJM 2015). However, these proposed classification systems differ in several aspects and yet no consensus has been established which genetic information is required for prognostication in AML today.Aims: 1) Test the prognostic value of a panel of molecular markers in addition to a cytogenetic score in a large cohort of AML patients. 2) Determine the proportion of patients with a suitable molecular marker for disease monitoring (MRD) applying molecular genetics.Patients and Methods: 867 de novo AML cases younger than 60 years were investigated (median age: 48 years, median follow up of 41 months). All patients were evaluated for karyotype, KMT2A-PTD, FLT3-ITD and in addition for mutation status of ASXL1, CEBPA, DNMT3A, NPM1, RUNX1 and TP53 according to the proposal by Grimwade et al. Blood 2016.Results: First, AML were classified according to the refined MRC cytogenetic classification with AML with t(15;17)/PML-RARA regarded as a separate group (n=89 (10%), 90% overall survival (OS) at 5 years). 89 cases (10%) were assigned to the favourable risk group (t(8;21)/RUNX1-RUNX1T1: n=42; inv(16)/t(16;16)/CBFB-MYH11: n=47), 570 (68%) to the intermediate risk group and 119 (14%) to the adverse risk group. OS at 5 years was 66%, 53% and 28%, respectively, and differed significantly between all four subgroups (for all comparisons p<0.001). Next, the following subgroups were separated: CEBPA double mutated (dm) cases (n=44 (5%); OS at 5 years: 83%), NPM1mut/FLT3-ITD- AML (n=181 (21%); OS at 5 years: 62%), and NPM1mut/FLT3-ITD+ AML (n=137 (16%); OS at 5 years: 47%; for all comparisons between these 3 groups p<0.001). Thus, prognosis of CEBPAdm cases was comparable to PML-RARA+ AML. OS in NPM1mut/FLT3-ITD- AML is comparable to CBF-leukemias. In NPM1mut AML no prognostic impact of DNMT3Amut was found. In all these 3 groups defined on molecular genetics no prognostic impact of additional karyotype information on OS was observed. Next, in the remaining cases of the cytogenetic intermediate risk group (n=209) the prognostic impact of mutations in ASXL1, DNMT3A, RUNX1, TP53, KMT2A-PTD and FLT3-ITD was evaluated. In multivariate Cox regression analysis mutations in TP53 (relative risk (RR): 3.5; p=0.04), ASXL1 (RR: 2.2, p=0.004), and FLT3-ITD (RR: 1.8; p=0.04) were independently associated with shorter OS. OS at 5 years was 25% in cases carrying at least one of these mutations compared to 54% in cases with none of these mutations (p=0.001). Within the adverse cytogenetic risk group cases with either a complex karyotype (n=27) or a KMT2A (n=25) or MECOM rearrangement (n=14) had the worst outcome compared to the remaining cases (OS at 5 yrs: 19% vs 54%, p=0.02). In the remaining cases the presence of at least one mutation in either ASXL1, TP53 or FLT3-ITD was associated with worse outcome (OS at 5 yrs: 33% vs 74%, p=0.04). Thus, AML with complex karyotype, KMT2A or MECOM rearrangements had the worst prognosis, while cases with adverse cytogenetics and at least one mutation in either ASXL1, TP53 or FLT3-ITD have a slightly better outcome which is comparable to AML with intermediate risk cytogenetics harbouring one of these mutations. OS in AML with adverse cytogenetics without mutations in ASXL1, TP53 or FLT3-ITD is not worse than in AML with intermediate cytogenetics also lacking these mutations.A fusion gene or a molecular mutation as target for MRD monitoring was present in 791 patients (91%) when all genes analysed were taken into account. If only markers showing prognostic relevance, i.e. fusion genes, mutations in NPM1, CEBPA, FLT3-ITD, ASXL1 and TP53 were considered a MRD marker was still available in 726 cases (84%).Conclusions: 1) In AML a prognostication system is feasible based on the identification of t(15;17)/PML-RARA, t(8;21)/RUNX1-RUNX1T1, inv(16)/t(16;16)/CBFB-MYH11, 11q23/KMT2A rearrangements, 3q26/MECOM rearrangements, complex karyotype, and mutation status of NPM1, CEBPA, ASXL1, TP53, and FLT3-ITD (figure 1). 2) The analysis of these parameters allows to identify an MRD marker in 84% of patients. 3) The analysis of additional genes may be required in a comprehensive AML work-up as soon as novel targeted treatment strategies will become available. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Non-Invasive Genetic Profiling Is Highly Applicable in Multiple Myeloma (MM) through Characterization of Circulating Tumor Cells (CTCs)

Chromosomal abnormalities (CA) play a pivotal role in predicting MM patients risk. Accordingly, well-defined CA have been associated with higher risk of transformation in smoldering MM (SMM), have been recently incorporated to the revised ISS, and may even guide treatment decisions; furthermore, because high-risk CA can be present in minor subclones at diagnosis it is recommended to reassess them at the time of relapse. Altogether, this implies that throughout their clinical course MM patients go through several bone marrow (BM) aspirates for cytogenetic risk assessment, and less invasive methods to screen for CA [ie. peripheral blood (PB) liquid biopsies] would be mostly welcomed providing they are fully comparable to its assessment in BM clonal plasma cells (PCs).Here, using the recently developed and sensitive next generation flow (NGF) method to monitor MRD in MM (median limit of detection, 3x10-6), we assessed the applicability of screening for CA in the PB, by determining the frequency of MGUS and MM patients with detectable CTCs and their respective absolute numbers. Using NGF, we detected CTCs in 22/37 (60%) MGUS cases, 9/12 (75%) SMM, 24/25 (96%) newly diagnosed MM, and 22/26 (85%) relapsed MM. Median numbers of CTCs/µL in the PB of MGUS, SMM, newly diagnosed and relapsed MM were 0.04, 0.1, 0.5 and 0.6, respectively. Thus, we noted highly significant differences (P<.001) between benign and malignant disease stages, with a 12.5-fold increment in CTCs/µL between MGUS and MM, and 5-fold between SMM and MM.By using whole exome sequencing, we reported recently that the concordance in CA between CTCs and BM clonal PCs was typically <50% due to the limited amount of DNA extracted from low numbers of CTCs, which required whole genome amplification. Accordingly, we investigated in 18 patient paired BM and PB (~5mL) samples (2 MGUS, 2 SMM and 14 MM) whether after NGF sorting and using the Affymetrix CytoScan HD (which requires significantly less DNA to provide molecular karyotypes), we could determine the pattern of CA in BM clonal PCs by screening that of CTCs. In 10 patients in which CTCs were successfully characterized, we obtained 100% concordance between BM clonal PCs and CTCs at the chromosome level (including trisomies of chromosomes 3, 5 and 21 which have been described to modulate the prognosis of patients with high-risk MM), for chromosomal arms, and for interstitial gains or losses (Panel A). Importantly, there was also 100% concordance with FISH performed on CD138+ BM clonal PCs for del(1p), +1q, del(13q) and del(17p) CA. In the remaining cases, ultra-low numbers of sorted CTCs (≤5.000) precluded their successful characterization, which could be readily overcome by using larger volumes of PB (ie. >5mL). Because the CytoScan HD does not assess the t(4;14), we included FGFR3 into the NGF panel of mAb in order to detect its surface expression in patients harboring the t(4;14). Similarly to that described above, we observed 100% concordance between FGFR3 expression in CTCs and the presence of t(4;14) in CD138+ BM clonal PCs, as confirmed by FISH (Panel B).Since gene expression profiling (GEP) has also been used to predict risk in MM, we investigated in 12 patient paired BM and PB samples whether after NGF sorting and using the Affymetrix Human Gene 2.0 ST Array, we could determine patients risk by screening the GEP of CTCs. Unsupervised analysis showed correct clustering between GEP of BM clonal PCs and CTCs in 9/12 cases, demonstrating that individual patient signatures typically outweighed GEP of MM clones located in different tissues (Panel C). Thus, CTCs could be used to predict risk according to well-established GEP signatures.In conclusion, by using sensitive NGF we showed that CTCs are present in 60% of MGUS and 87% of MM, which unravels high feasibility for liquid biopsying these patients. By also using NGF to sort CTCs, as well as commercially available, cost-effective and standardized arrays to determine CA and gene expression profiles, we demonstrated for the first time that the genetic features of BM clonal PCs can be determined with great accuracy by studying CTCs. Altogether, this combined approach could offer to many MM patients a less invasive cytogenetic characterization at certain stages of their clinical course. [Display omitted] DisclosuresPaiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

“Serpentine-like syndrome”–A very rare multiple malformation syndrome characterised by brachioesophagus and vertebral anomalies

“Serpentine-like syndrome” is a severe and rare association of multiple congenital malformations, characterised by brachioesophagus, secondary intrathoracic stomach, and vertebral anomalies. Other associated anomalies have been described, such as malposition and herniation of abdominal organs.We report the natural history of a baby girl born at 29 weeks of gestation with intra uterine growth restriction, short neck, large rachischisis from cervical to thoracic spine, a very short oesophagus, thoracic stomach associated with a midline diaphragmatic hernia, malrotated gut and median cleft lip. Most of these anomalies were detected antenatally. Molecular karyotype was normal. She died at age 12 days.To our knowledge, the present patient represents the 8th report of a case of “Serpentine-like syndrome”. Brachioesophagus and congenital vertebral anomalies, in particular rachischisis, are the cardinal features of this condition. All reported cases have been sporadic and the cause is still unknown. We believe that the specificity of the presentation as well as the similarities between available descriptions of patients suggests a common, yet to identify, molecular cause, possibly involving a developmental “toolkit”/homeobox gene or related pathways.