Abnormal Ploidy Research Articles

Single Cell Mutation Analysis Delineates Clonal Architecture in Leukemic Transformation of Myeloproliferative Neoplasms

Introduction. Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells that include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. 10-20% of MPN pts transform to secondary acute myeloid leukemia (sAML), unresponsive to conventional therapy and associated with dismal outcome (Dunbar A, 2020). In addition to somatic driver mutations affecting JAK2, CALR or MPL, several additional variants are harbored by MPN pts inb chronic phase, and a restricted set of them were associated with risk of leukemic evolution (Vannucchi AM, Leukemia 2013; Tefferi A, Blood ASdv 2016) However, the molecular mechanisms underlying leukemic transformation remain largely unknown. Although bulk next generation sequencing (NGS) highlights the overall mutation landscape, it cannot distinguish which mutations occur in the same clone(s), nor elucidate the order of mutations or resolve clonal complexity. Conversely, single-cell sequencing (SCS) might allow to resolve clonal heterogeneity and reconstruct clonal phylogenies at each disease phase (Parenti, NPJ Prec Onc 2021)).Aim: To delineate the clonal landscape of sAML, we performed single-cell mutational profiling in 10 pts with MPNs who progressed to sAML.Methods. There were 2 set of samples/approaches: (i) 15 paired samples (chronic (CP)/blast phase (BP)) from 7 pts were analyzed using the Mission-Bio Tapestri platform and the Myeloid panel in order to target SNVs and indels across 45 myeloid genes with 312 amplicons. In one pt we also analyzed an intermediate phase corresponding to progression from PV to PPV-MF before BP development. (ii) 7 further paired CD34+ samples from 3 pts were analyzed using a 239-amplicon custom panel including 29 genes frequently mutated in myeloid neoplasms. SCS libraries were sequenced on Illumina Novaseq. Data were processed by using Mission Bio's Tapestri Pipeline and analyzed with Mission Bio's Tapestri Insights software package and R software. CNV analysis was performed by using an integrated pipeline for multiomics analysis (Mosaic, Mission Bio)Results. (i) A total of 78,354 single cells were sequenced (average 5,223) using Tapestri Myeloid panel, with an average of 28,303 reads per cell and coverage of 97X. SCS was able to identify 17 low-frequency variants not detected in bulk analysis; however, it failed to discriminate homopolymeric regions including the ASXL1 G646Wfs*12. (ii) A total of 25,417 single cells were sequenced (average 3636) using a custom panel, with a coverage of 186X and an average Allele Dropout Rate of 8.6%. This panel was able to identify ASXL1 G646Wfs*12 variant. Overall, we found a significant correlation of variant allele frequency (VAF) measured by bulk and SCs approach (R =0.84, p<.0001). Epigenetic variants (i.e. ASXL1, TET2, EZH2) account for around half of the mutations and affect a large fraction of CP cells (3 representative samples in Fig.1). In 8/10 pts, leukemic clone emerged from a driver mutation-positive cells (JAK2V617F n=4; CALR Type1 n=4). In all pts we are able to identify at least 3 mutated clones and in 7 pts the dynamics of the clones allowed to identify the one(s) responsible for evolution to sAML. In 7/10 pts, the leukemic clones were already detectable at low frequency (<2%) at CP and became dominant in BP; these low-frequency clones were missed by bulk sequencing. Furthermore, SCS revealed acquisition of 3 mutually exclusive mutations in RAS pathway in one pt: two NRAS mutations and a KRAS mutation. Copy number variation (CNV) could be assessed in 4 pts. Of note, during progression to sAML, we found single cells with amplification of ETV6 (>20 copies in 2 pts), NRAS (8 copies in 2pts) and BRAF (8 copies in 2pts). Other subclonal ploidy abnormalities were also observed in RUNX1, EZH2, U2AF1 and ZRSR2 (5-18 copies).Conclusions. Together, these data suggest that MPN present a complex clonal combination evolving over time. SCS allows to resolve this milieu, that is largely missed by conventional bulk NGS, in particularly SCS identifies rare leukemia-driving clones that were already present in chronic phase and describes their dynamics during leukemic progression. Leukemic transformation after MPN is a highly heterogeneous process with mutations and CNVs acquired in different genes and different clones. Overall, our findings provide further insights into the pathogenesis of AML transformation of MPN.Supported by AIRC, Mynerva project no.21267 [Display omitted] DisclosuresVannucchi: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Accurate Prediction of Chromosome-Level CNVs from Targeted NGS

Background: Aneuploidy and large-scale Copy Number Variations (CNVs) are prominent features of cancer cells. While Fluorescence in situ hybridization (FISH) and conventional cytogenetics (CC) are the gold standard for detecting aneuploidy and CNVs, NGS-based assays are currently used for high-resolution detection of copy number alterations assessing the whole genome. However, although an increasing number of NGS-based tools have been developed for detecting aneuploidy or CNVs from whole genome or exome sequencing data, only a limited number of options are available for targeted gene panels. Despite mechanisms provided to establish normal profiles for a specific panel, the accuracy of these tools at the chromosome level suffer when only a small number of regions are targeted on each chromosome. Here we leveraged on a custom amplicon based NGS assay designed to detect somatic alterations (SNVs and indels) in 297 hematological cancer relevant genes, previously validated in our clinical laboratory. We introduce a simple approach to accurately predict chromosome-level CNVs such as monosomy and trisomy for a targeted gene panel, commonly used in a clinical setting.Methods: Mutation profiles, including SNVs, INDELs, and structural changes, were interrogated with an in-house bioinformatics pipeline that utilized PureCN and CNVkit algorithms to detect structural changes. The first step consists of finding optimal panel-specific decision thresholds for gains and losses at the gene level. This step was performed using an independent set of 1,314 clinical samples sequenced with the NeoType® Heme assay developed by NeoGenomics Laboratories, Inc. for which at least one FISH test was performed in addition to the sequencing. Three genes (ATM, TP53, and NF1) were used to find optimal decision thresholds based on the FISH result for these markers. These thresholds are used afterward to predict a gain or a loss for any other gene in the panel. The second step consists of predicting the chromosome-level gain or loss based on the individual predictions at the gene level by simply observing the frequency of targeted genes on the corresponding chromosome predicted as either gained or lost by the first step approach. The 19, 7, and 18 targeted genes in the NGS panel (Table 1) were respectively used to predict monosomy 7, trisomy 8, and trisomy 12 in a second set of over 7,000 clinical samples with known ploidy for chromosomes with clinically relevant ploidy abnormalities in hematological malignancies.Results: Evaluation of the first stage gene-level CNV prediction on 1,314 clinical samples shows a concordance rate of 97.95% between NGS and FISH results on ATM, TP53, and NF1. When we evaluated the second stage chromosome-level CNV prediction in clinical samples sequenced using the same targeted panel and assessed by FISH for chromosome-level variation on chromosomes 7, 8 and 12 (Table 1), a heatmap of the predicted Log 2 ratios for each sample and targeted gene from the first step shows a clear distinctive signal between aneuploidy and diploid samples (Figure 1). At the chromosome level, the concordance rate between the final prediction and the FISH results is consistently observed above 93% (Table 2). Roughly 50% of the 12, 78, and 40 discordant calls for monosomy 7, trisomy 8, and trisomy 12, respectively captured by FISH but not by NGS can be explained by low tumor content (less than 20%) in the tested samples. The concordance rate between NGS and FISH is consistently observed above 96% when leaving these samples aside. Note that results in Table 2 are obtained using all samples to decide the optimal decision threshold for the chromosome-level prediction, but are found identical when using a leave-one-out evaluation procedure, and nearly identical when using a repeated cross-validation procedure.Conclusion: This study demonstrates that chromosome-level CNVs can be accurately predicted in hematologic malignancies even when the number of targeted genes on a given chromosome is low. Despite the simplicity of the approach, the two stages bioinformatics pipeline based on an ensemble method allowed us to gain between 8% and 46% accuracy compared to relying only on the prediction of a single tool like PureCN. Samples with low tumor content remain, however, a difficult case to tackle with bulk NGS as it is difficult to distinguish a CNV from the natural variability of the sequencing coverage. [Display omitted] DisclosuresNam: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.

Exploration of the Cytoplasmic Function of Abnormally Fertilized Embryos via Novel Pronuclear-Stage Cytoplasmic Transfer.

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory “aged” oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.

Experience analysing over 190,000 embryo trophectoderm biopsies using a novel FAST-SeqS preimplantation genetic testing assay

Research questionIs FAST-SeqS an accurate methodology for preimplantation genetic testing for whole-chromosome aneuploidy (PGT-A)? What additional types of chromosomal abnormalities can be assessed? What are the observed aneuploidy rates in a large clinical cohort? DesignFAST-SeqS, a next-generation sequencing (NGS)-based assay amplifying genome-wide LINE1 repetitive sequences, was validated using reference samples. Sensitivity and specificity were calculated. Clinically derived trophectoderm biopsies submitted for PGT-A were assessed, and aneuploidy and mosaicism rates among biopsies were determined. Clinician-provided outcome rates were calculated. ResultsSensitivity and specificity were over 95% for all aneuploidy types tested in the validation. Comparison of FAST-SeqS with VeriSeq showed high concordance (98.5%). Among embryos with actionable results (n = 182,827), 46.2% were aneuploid. Whole-chromosome aneuploidies were most observed (72.9% without or 8.7% with a segmental aneuploidy), with rates increasing with egg age; segmental aneuploidy rates did not. Segmental aneuploidy (n = 20,557) was observed on all chromosomes (most commonly deletions), with frequencies associated with chromosome length. Mosaic-only abnormalities constituted 10.1% (n = 3862/38145) of samples. Abnormal ploidy constituted 1.8% (n = 2370/128,991) of samples, triploidy being the most common (73.6%). Across 3297 frozen embryo transfers, the mean clinical pregnancy rate was 62% (range 38–80%); the mean combined ongoing pregnancy and live birth rate was 57% (range 38–72%). ConclusionFAST-SeqS is a clinically reliable and scalable method for PGT-A, is comparable to whole-genome amplification-based platforms, and detects additional information related to ploidy using SNP analysis. Results suggest ongoing benefit of PGT-A using FAST-SeqS, consistent with other platforms.

Appearance, fate and utilization of abnormal porcine embryos produced by in vitro maturation and fertilization

In vitro production (IVP) including in vitro maturation (IVM) and fertilization (IVF) is now an important technology for obtaining live piglets. However, there are still two significant obstacles to the efficient production of viable porcine embryos: (1) polyspermy and (2) fertilization of oocytes arrested at the immature stage. These phenomena relate to production of embryos with abnormal ploidy (polyploidy). To avoid these problems, careful selection of mature oocytes for IVF, and regular monitoring of normal and abnormal fertilization (polyspermy and/or lack of male pronucleus formation) are very important. In our recent studies, however, we have confirmed that some oocytes with abnormal ploidy after polyspermy can develop into diploid embryos with potentially normal developmental ability. The mechanism by which such fertilized polyploid oocytes develop to a normal state during embryo development is still not well understood. Attempts to clarify this mechanism would hopefully reveal data that are very useful for not only IVP but also other technologies such as the production of transgenic or cloned animals using IVM oocytes, including other species, also for human reproductive manipulation. In this review, we focus on studies of normality of IVM oocytes and ploidy of IW embryos, and try to suggest practical ways of solving the problems mentioned above in pigs.

Ploidy Levels and Fitness-Related Traits in Purebreds and Hybrids Originating from Sterlet (Acipenser ruthenus) and Unusual Ploidy Levels of Siberian Sturgeon (A. baerii).

The present study aimed to investigate and compare fitness-related traits and ploidy levels of purebreds and hybrids produced from sturgeon broodstock with both normal and abnormal ploidy levels. We used diploid Acipenser ruthenus and tetraploid A. baerii males and females to produce purebreds and reciprocal hybrids of normal ploidy levels. Likewise, we used diploid A. ruthenus and tetraploid A. baerii females mated to pentaploid and hexaploid A. baerii males to produce hybrids of abnormal ploidy levels. Fertilization of ova of A. ruthenus and A. baerii of normal ploidy with the sperm of pentaploid and hexaploid A. baerii produced fully viable progeny with ploidy levels that were intermediate between those of the parents as was also found in crosses of purebreds and reciprocal hybrids of normal ploidy levels. The A. ruthenus × pentaploid A. baerii and A. ruthenus × hexaploid A. baerii hybrids did not survive after 22 days post-hatch (dph). Mean body weight and cumulative survival were periodically checked at seven-time intervals. The recorded values of mean body weight were significantly higher in A. baerii × pentaploid A. baerii hybrids than other groups at three sampling points (160, 252 and 330 dph). In contrast, the highest cumulative survival was observed in A. baerii × A. ruthenus hybrids at all sampling points (14.47 ± 5.70 at 497 dph). Overall, most of the studied sturgeon hybrids displayed higher mean BW and cumulative survival compared to the purebreds. The utilization of sturgeon hybrids should be restricted to aquaculture purposes because they can pose a significant genetic threat to native populations through ecological interactions.

Rapamycin treatment correlates changes in primary cilia expression with cell cycle regulation in epithelial cells

Primary cilia are sensory organelles that regulate cell cycle and signaling pathways. In addition to its association with cancer, dysfunction of primary cilia is responsible for the pathogenesis of polycystic kidney disease (PKD) and other ciliopathies. Because the association between cilia formation or length and cell cycle or division is poorly understood, we here evaluated their correlation in this study. Using Spectral Karyotyping (SKY) technique, we showed that PKD and the cancer/tumorigenic epithelial cells PC3, DU145, and NL20-TA were associated with abnormal ploidy. We also showed that PKD and the cancer epithelia were highly proliferative. Importantly, the cancer epithelial cells had a reduction in the presence and/or length of primary cilia relative to the normal kidney (NK) cells. We then used rapamycin to restore the expression and length of primary cilia in these cells. Our subsequent analyses indicated that both the presence and length of primary cilia were inversely correlated with cell proliferation. Collectively, our data suggest that restoring the presence and/or length of primary cilia may serve as a novel approach to inhibit cancer cell proliferation.

Clinical value of quantitative detection of DNA aneuploidy in the diagnosis and treatment of cervical lesions in middle-aged and senile women

Objective To investigate the clinical value of quantitative detection of DNA aneuploidy in the diagnosis and treatment of cervical lesions in middle-aged and senile women. Methods A total of 1 404 middle-aged and elderly women who underwent screening for early cervical lesions were retrospectively studied.Patients were divided into the two groups: the 40-49 years old group(n=897)and the 50-78 years old group(n=507). Cervical lesions were screened by DNA ploidy analysis and the results were compared with those screened by liquid-based cytology, colposcopy and high-risk human papillomavirus(HPV). Results The positive detection rate of HPV by DNA ploidy analysis was 54.4%(764/1 404). Of 1 404 patients, HPV16/18 infection accounted for 21.3%(299/1 404). The detection rate of heteroploid cells was 50.92%(715/1 404). There was a significant positive correlation between HPV infection type and cervical epithelial cell ploidy changes(r=870, P=0.001). The detection rate of HPV by liquid-based cytology was 45.08%, which was lower than that by DNA aneuploidy(χ2=9.594, P=0.002). The differences in the incidences of low-grade squamous intraepithelial lesion(LSIL)and high-grade squamous intraepithelial lesion(HSIL)and above categories of lesions were statistically significant(χ2=289.598, P=0.000)between patients with and without DNA aneuploidy.The statistically significant differences were found between the 40-49 years old group and 50-78 years old group(P<0.05)in the occurrence of abnormal DNA ploidy cells, HPV infection rate, the proportion of LSIL, HSIL and above categories of lesions. Conclusions Compared with the conventional cytology, DNA aneuploidy quantitative detection has higher sensitivity and better specificity, and has no significant difference from the high-risk HPV detection.It can be used as one of methods for screening cervical lesions in middle-aged and elderly women, especially those with high-risk HPV infection. Key words: DNA aneuploid; Uterine cervical neoplasms; Papillomavirus infections

Mechanical shock during egg de-adhesion and post-ovulatory ageing contribute to spontaneous autopolyploidy in white sturgeon culture (Acipenser transmontanus)

Spontaneous autopolyploidy, a 1.5x increase in genome size resulting from second polar body retention after fertilization, has been documented in cultured sturgeons, with the proportion of spontaneous autopolyploid progeny ranging widely among maternal families. Sturgeon farms wish to reduce the number of spontaneous autopolyploids because their progeny, when crossed with a normal ploidy parent, exhibit intermediate ploidies, resulting in reproductive abnormalities. However, there is limited knowledge of the causes of the second polar body retention in sturgeon culture. In this study, we report the results of experiments performed from 2015 to 2019 aimed at identifying the sources of spontaneous autopolyploidy in white sturgeon (Acipenser transmontanus) culture. In collaboration with several sturgeon farms, we tested whether post-ovulatory ageing, mechanical shock during egg de-adhesion, and the combined effects of both factors increased spontaneous autopolyploidy. To test the effect of post-ovulatory ageing, eggs were collected from females and either fertilized at the industry normative time (2–5 h post-oviposition) or stored in ovarian fluid at 15 °C for 6–8 h before fertilization. To test the effect of mechanical shock, eggs were collected, fertilized 2–5 h post-oviposition and exposed to either gentle or vigorous mixing during the 60 min de-adhesion treatment. Results from this work reveal that post-ovulatory ageing does increase the incidence of spontaneous autopolyploidy in some females, but overall the proportions produced were low (range 0–15%). Proportions of spontaneous autopolyploids in eggs exposed to vigorous mixing were also variable (1–92%) but significantly higher in 75% of the females when compared to their respective controls or gentle mixing treatments, indicating that mechanical shock during egg de-adhesion is likely the primary cause of spontaneous autopolyploidy. To our knowledge this is the first study to document mechanical shock to eggs during de-adhesion as a cause of abnormal ploidy in cultured fishes. We observed high variability in egg quality among females and a significant relationship between embryo mortality and the incidence of spontaneous autopolyploidy when eggs were exposed to mechanical shock. Repeated spawning of a female that produced a high proportion of spontaneous autopolyploids provided preliminary evidence that genetic background may influence the likelihood that a female's eggs will be prone to second polar body retention when subjected to mechanical shock. Although spontaneous autopolyploidy in sturgeon culture will likely never be eliminated entirely, we provide practical recommendations to sturgeon producers to reduce its incidence in a production setting.

Impact of technology changes on the identification and testing of abnormally fertilized embryos

Introduction Blastocyst biopsy has been the standard practice at Genea for the last 15 years. In contrast, the technology used for embryo culture and the analysis of biopsied tissue samples has changed markedly. Most recently, the introduction of continuous culture with imaging capabilities (Geri, Genea Biomedx) and Next Generation Sequencing (NGS; Illumina VeriSeq) have combined to have a positive impact on our ability to detect abnormal ploidy states. Materials & Methods To control for staggered implementation times of new technologies, the dataset used in this study was confined to cycles undergoing PGT-A at our main clinic. IVF/ICSI cycle and PGT-A data since the start of 2014 were retrospectively analyzed, representing a period of approximately two years before and after the introduction of NGS and Geri. The rates of abnormal fertilization events identified by fertilization checks and by PGT, as well as clinical outcomes, were assessed. Results Implementation of PGT-A by NGS improved detection rates for triploid 69XXY and 69XYY embryos, with a total of 56 identified. However, the trend of increased triploid detection is now reversing, which correlates with improved detection of 3PN fertilization events due to time-lapse imaging. Although 50% of embryos in the study group were derived from ICSI, 70% of triploid results were from IVF insemination. Roll out of continuous culture with time-lapse capabilities has also correlated with an increase in the proportion of monopronuclear (1PN) embryos detected at fertilization check, with an overall rate of 5%. Increased uptake of PGT-A, combined with better education regarding the options for testing of 1PN embryos, has resulted in increased detection of abnormal 1PN blastocysts. 1PNs returning a PGT-A result of NAD XY were considered normal, whereas 70 apparently balanced NAD XX embryos were further tested by STR analysis. Abnormal fertilization events were confirmed for ten of these, seven of which had maternal only and three paternal only contributions. Although 65% of tested 1PN embryos were derived from IVF insemination, all except one (maternal only) of the verified abnormal fertilization events were ICSI inseminations. Transfer of 39 1PN embryos with confirmed biparental inheritance has resulted in 18 biochemical pregnancies, 15 of which proceeded to a fetal heartbeat, with 13 live births to date. Conclusions IVF fertilized embryos that develop to a biopsiable blastocyst appear to have a very low risk of being true 1PNs, but have an increased chance of being triploid relative to ICSI. In contrast, a significant proportion of tested 1PN ICSI embryos were confirmed to have monoparental inheritance. Given that paternal only 1PNs derived from a Y sperm will not develop, our results suggest that monoparental ICSI embryos arise with equal frequency from maternal only and paternal only events. Overall, our experience indicates that implementation of continuous culture with imaging capabilities, combined with encouraging patients to consider the option of PGT, can decrease the risk of transferring an embryo with abnormal ploidy status, while also improving overall cycle success rates by making many 1PN embryos available for clinical use.

A comparison of methods for determining ploidy in white sturgeon (Acipenser transmontanus)

Release of sturgeon with abnormal ploidy into the wild may result in reduced fitness due to lowered fertility in the F2 and subsequent generations. Further, there is evidence that ploidy affects reproductive development and caviar yield. Therefore, the ability to accurately characterize the ploidy of white sturgeon (Acipenser transmontanus) is essential for both commercial and conservation aquaculture. This study compares nuclear volume and whole erythrocyte long-axis lengths obtained using Coulter counter and blood smears, respectively, from captive white sturgeon from populations originating in California and Idaho. We examine which method provides the most accurate, time efficient and cost-effective characterization of ploidy in this species. Results from Coulter counter and blood smears were compared to results from flow cytometry, the gold standard for genome size analysis. Previous work suggests that blood smears can distinguish between 8N (diploid) and 12N (triploid) sturgeon, but further analysis is required to see if this method can also be used to identify 10N fish and to provide robust evidence of its utility in 8N and 12N fish across populations. In this study, we demonstrated that the Coulter counter had 100% agreement with flow cytometry in ploidy assignment, while blood smears vary in their accuracy based on population. Blood smears showed a high degree of overlap in erythrocyte long-axis length between 8N and 10N individuals as well as some overlap between 10N and 12N individuals in the California fish, and a high degree of overlap between 8N and 12N individuals in the Idaho fish. Although blood smears are time-intensive and vary in their ploidy assignment accuracy, they are a low-cost technique and as such may have some utility for caviar farms attempting to identify 12N individuals in a small number of broodstock. By comparing the accuracy, efficiency and cost of these three methods, sturgeon farmers and conservation hatcheries will be able to choose the best method for their needs in determining the ploidy of their fish. We determined that Coulter counter is equally accurate to flow cytometry and is also the most time efficient method for ploidy determination in white sturgeon.

Chromosome screening using culture medium of embryos fertilised in vitro: a pilot clinical study

BackgroundPrevious studies from this as well as other research groups suggested that non-invasive chromosome screening (NICS) with embryo culture medium can be used to identify chromosomal ploidy and chromosomal abnormalities. We here report a series of clinical cases utilizing the technology.MethodsA total of 45 couples underwent in vitro fertilisation during a period between February 2016 and February 2017. Karyotyping revealed normal chromosomes in both partners in 23 couples, and chromosomal rearrangements in at least one partner in 22 couples. Intracytoplasmic sperm injection (ICSI) was used for fertilization. NICS was carried out using embryo culture medium at the blastocyst stage via multiple annealing and looping-based amplification cycles, whole-genome amplification and next-generation sequencing.ResultsA total of 413 embryos were obtained; 170 blastocysts were subjected to NICS. The screening showed euploidy in 79 embryos, aneuploidy in 52 embryos, and mosaic ploidy for 33 embryos. The rate of euploidy was comparable in couples with normal karyotype (50.7%; 38/75) vs. chromosomal rearrangement (43.2%; 41/95). A total of 52 euploid embryos (50 oocyte retrieval cycles) were transferred in 43 women. Biochemical pregnancy rate was 72.0% (36/50). Clinical pregnancy rate was 58.0% (29/50). The rate of spontaneous miscarriage was 3/29 (none with chromosomal aneuploidy). A total of 27 healthy babies were delivered.ConclusionsNICS could identify embryo chromosomal abnormalities in couples either with or without chromosomal rearrangement, with satisfying clinical outcomes.

Chromosomal instability-induced senescence potentiates cell non-autonomous tumourigenic effects

Chromosomal instability (CIN), a high rate of chromosome loss or gain, is often associated with poor prognosis and drug resistance in cancers. Aneuploid, including near-polyploid, cells contain an abnormal number of chromosomes and exhibit CIN. The post-mitotic cell fates following generation of different degrees of chromosome mis-segregation and aneuploidy are unclear. Here we used aneuploidy inducers, nocodazole and reversine, to create different levels of aneuploidy. A higher extent of aneuploid and near-polyploid cells in a given population led to senescence. This was in contrast to cells with relatively lower levels of abnormal ploidy that continued to proliferate. Our findings revealed that senescence was accompanied by DNA damage and robust p53 activation. These senescent cells acquired the senescence-associated secretory phenotype (SASP). Depletion of p53 reduced the number of senescent cells with concomitant increase in cells undergoing DNA replication. Characterisation of these SASP factors demonstrated that they conferred paracrine pro-tumourigenic effects such as invasion, migration and angiogenesis both in vitro and in vivo. Finally, a correlation between increased aneuploidy and senescence was observed at the invasive front in breast carcinomas. Our findings demonstrate functional non-equivalence of discernable aneuploidies on tumourigenesis and suggest a cell non-autonomous mechanism by which aneuploidy-induced senescent cells and SASP can affect the tumour microenvironment to promote tumour progression.

Genome doubling shapes the evolution and prognosis of advanced cancers.

Ploidy abnormalities are a hallmark of human cancers, but their impact on the evolution and outcomes of cancers is unknown. Here, we identified whole-genome doubling (WGD) in the tumors of nearly 30% of 9,692 prospectively sequenced advanced cancer patients. WGD varied by tumor lineage and molecular subtype and arose early in the pathogenesis of affected cancers after an antecedent transforming driver mutation. While associated with TP53 mutations, 46% of all WGD arose in TP53-wildtype tumors and, in such cases, was associated with an E2F-mediated G1 arrest defect, though neither aberration was obligate in WGD tumors. The variability of WGD across cancer types can be explained in part by cancer cell proliferation rates. WGD predicted for increased risk of death in tumors pan-cancer, a negative impact independent of established clinical prognostic factors in multiple cancer types including KRAS-mutant colorectal cancers and estrogen receptor-positive breast cancers. WGD is one of the most common genomic events in cancer and is a macro-evolutionary event associated with poor prognosis across cancer types.

Neuroblastoma: clinical and biological approach to risk stratification and treatment.

Neuroblastoma is the most common extra-cranial solid tumor of childhood and the most common in the first year of life. It is a unique malignancy in that infants often present with either localized or metastatic disease that can spontaneously regress without intervention while older children can succumb to the disease after months to years of arduous therapy. Given this wide range of outcomes, the International Neuroblastoma Risk Group was created to stratify patients based on presenting characteristics and tumor biology in order to guide intensity of treatment strategies. The goal has been to decrease therapy for low-risk patients to avoid long-term complications while augmenting and targeting therapies for high-risk patients to improve overall survival. The international risk stratification depends on age, stage, histology, MYCN gene amplification status, tumor cell ploidy and segmental chromosomal abnormalities. Treatment for asymptomatic low-risk patients with an estimated survival of > 98% is often observation or surgical resection alone, whereas intermediate-risk patients with an estimated survival of > 90% require moderate doses of response-adjusted chemotherapy along with resection. High-risk patients undergo multiple cycles of combination chemotherapy before surgery, followed by consolidation with myeloablative autologous hematopoietic stem cell transplantation and local radiation and finally immunotherapy with differentiation therapy as maintenance phase. With this approach, outcome for patients with neuroblastoma has improved, as the field continues to expand efforts in more targeted therapies for high-risk patients.

Assessment of DNA Ploidy in the ProMisE molecular subgroups of endometrial cancer

ObjectiveWe sought to determine whether DNA ploidy correlates with the four molecular subgroups of endometrial carcinoma (EC) as determined using ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer). Methods90 cases of EC previously characterized by clinicopathological parameters, outcomes, and ProMisE molecular subgroup (POLE EDM, MMR-D, p53 wt or p53 abn) were assessed for DNA ploidy using image cytometry. Associations of ploidy with traditional clinicopathological parameters were also tested. ResultsAbnormal DNA ploidy status differed amongst the ProMisE groups (p<0.001) and was found in 80.9% (17/21) of p53 abn, 37.0% (10/27) of p53 wt, 28.6% (4/14) of POLE EDM and 14.3% (4/28) of MMR-D. Abnormal DNA content was significantly associated with lower BMI (p=0.034) and grade 3 tumors (p=0.001). In the entire cohort, abnormal DNA content was significantly associated with worse progression free survival (p=0.0094) but not disease specific survival (p=0.249) or overall survival (p=0.187). When examining ploidy within each of the ProMisE groups, abnormal DNA content correlated with worse overall survival (p=0.041) and progression free survival (p=0.011) in the MMR-D group. No statistically significant relationship was seen in the remaining 3 groups. ConclusionAbnormal DNA ploidy status did correlate with the molecular subgroups of EC; abnormal DNA content was seen in the large majority of p53 abn cases. Abnormal ploidy however was also seen in smaller numbers in the p53 wt, POLE EDM and MMR-D groups; therefore abnormal DNA content was not a specific marker for any one molecular group. The addition of ploidy to the ProMisE molecular categories conferred additional prognostic value within the MMR-D group, which merits further study.

Porcine Cloned Embryos Reconstructed with the Cell Nuclei of Tetraploid M-phase Fibroblast Cells Can Restore Normal Diploidy at the Blastocyst Stage

ABSTRACTThe cell cycle of donor cells as a major factor that affects cloning efficiency remains debatable. G2/M phase cells as a donor can successfully produce cloned animals, but a minimal amount is known regarding nuclear remodeling events. In this study, porcine fetal fibroblasts (PFFs) were carefully synchronized at G1 or M phase as donor cells. Most of the cloned embryos reconstructed from PFFs at G1 (G1-embryos) or M (M-embryos) phase formed a pronucleus-like nucleus (PN) within 6-h post fusion (hpf), but the M-embryos formed PN earlier than the G1-embryos did. Moreover, 77.4% of the M-embryos formed two PNs, whereas the G1-embryos formed a single PN. The rate of extrusion of polar body-like structures by the M-embryos was significantly lower than that extruded by the G1-embryos (26.3% vs. 37.1%, P < 0.05), and DNA synthesis in most embryos in both groups was initiated at 9–12 hpf. Most of the M-embryos were octoploid before the first cleavage. Furthermore, 81.25% of the blastomeres of blastocysts developed from the M-embryos showed abnormal ploidy compared with those developed from the G1-embryos (22.55%). However, some of the blastomeres remained diploid in all the M-embryos tested. A portion of the blastomeres restored normal diploidy in some of the M-embryos at the blastocyst stage. This finding provides an explanation for M-embryos developing to term.

Microarray-based tumor ploidy and its association with lymph node involvement in bladder cancer.

e16012Background: Abnormal ploidy (or aneuploidy), defined as more or less than two full sets of chromosomes per cell, is often observed in tumor tissues. Aneuploidy and lymph node involvement (LNI...

Abstract P4-04-02: Amplification of chromosome 9p24 targeting PD-L1 and JAK2 correlates with altered tumor microenvironment expression profiles in triple negative breast cancer

Abstract Introduction: In a pilot study, we recently described amplification of chromosome 9p24 encoding PD-L1, PD-L2, and JAK2 (the PDJ amplicon) in 29% of early stage triple negative breast cancers (TNBC). Amplification was associated with poor DFS and OS. The impact of this amplicon on immune function is not known. Methods: Fresh frozen tumor samples from 36 subjects with newly-diagnosed TNBC (stages I-III) were evaluated for copy number aberrations using DNA-based flow cytometry to enrich nuclei based on ploidy status (tetraploidy or aneuploidy), followed by oligonucleotide CGH arrays. Targeted immune expression profiling was performed using the Nanostring PanCancer Immune Profiling Panel (n=770 genes). Results: 23 samples had measurable abnormal ploidy (evidence of sufficient tumor content) for CGH analysis; the remaining samples were omitted from analysis. High level (log2ratio &amp;gt;1) amplification was detected in 6/23 (26.1%) of TNBCs, and low-level copy number gain (CN log2ratio &amp;gt;0 and&amp;lt;1) was detected in 3/23 (13.0%). The remaining samples had no copy number change (n=9) or low level copy number loss (CN log2ratio&amp;gt;-1 and &amp;lt;0, n=5). Increased RNA expression of STAT1, STAT2, STAT3, and STAT6 was associated with PDJ amplification, as well as upregulation of the immune regulatory genes CD47 and IDO1. Conclusion: Amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in a significant proportion of TNBCs. This amplicon is associated with altered gene expression of the tumor microenvironment. Citation Format: Anderson KS, Andreozzi M, Pockaj BA, Hopper M, McCullough AE, Barrett MT. Amplification of chromosome 9p24 targeting PD-L1 and JAK2 correlates with altered tumor microenvironment expression profiles in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-02.

Mir-661: A key Factor in Embryo-Maternal dialog With Potential Clinical Application to Predict Implantation Outcome?

Mir-661: A key Factor in Embryo-Maternal dialog With Potential Clinical Application to Predict Implantation Outcome?