Brynn Levy

Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions

DNA damage checkpoint genes, such as p53, are frequently mutated in human cancer, but the selective pressure for their inactivation remains elusive. We analysed a panel of human lung hyperplasias, all of which retained wild-type p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and Chk2 phosphorylation, p53 accumulation, focal staining of p53 binding protein 1 (53BP1) and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). We propose that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability and selective pressure for p53 mutations.

Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci.

This study aims to develop a noninvasive prenatal test on the basis of the analysis of cell‐free DNA in maternal blood to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y.

In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial

OBJECTIVE To determine whether performing comprehensive chromosome screening (CCS) and transferring a single euploid blastocyst can result in an ongoing pregnancy rate that is equivalent to transferring two untested blastocysts while reducing the risk of multiple gestation. DESIGN Randomized, noninferiority trial. SETTING Academic center for reproductive medicine. PATIENT(S) Infertile couples (n = 205) with a female partner less than 43 years old having a serum anti-Müllerian hormone level ≥ 1.2 ng/mL and day 3 FSH <12 IU/L. INTERVENTION(S) Randomization occurred when at least two blastocysts were suitable for trophectoderm biopsy. The study group (n = 89) had all viable blastocysts biopsied for real-time, polymerase chain reaction-based CCS and single euploid blastocyst transfer. The control group (n = 86) had their two best-quality, untested blastocysts transferred. MAIN OUTCOME MEASURE(S) The ongoing pregnancy rate to ≥ 24 weeks (primary outcome) and the multiple gestation rate. RESULT(S) The ongoing pregnancy rate per randomized patient after the first ET was similar between groups (60.7% after single euploid blastocyst transfer vs. 65.1% after untested two-blastocyst transfer; relative risk [RR], 0.9; 95% confidence interval [CI], 0.7-1.2). A difference of greater than 20% in favor of two-blastocyst transfer was excluded. The risk of multiple gestation was reduced after single euploid blastocyst transfer (53.4% to 0%), and patients were nearly twice as likely to have an ongoing singleton pregnancy (60.7% vs. 33.7%; RR, 1.8; 95% CI, 1.3-2.5). CONCLUSION(S) In women ≤ 42 years old, transferring a single euploid blastocyst results in ongoing pregnancy rates that are the same as transferring two untested blastocysts while dramatically reducing the risk of twins.

First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy.

Abstract Objective: To develop a preimplantation genetic diagnosis (PGD) protocol that allows any form of chromosome imbalance to be detected. Design: Case report employing a method based on whole-genome amplification and comparative genomic hybridization (CGH). Setting: Clinical IVF laboratory. Patient(s): A 40-year-old IVF patient. Intervention(s): Polar body and blastomere biopsy. Main Outcome Measure(s): Detection of aneuploidy. Result(s): Chromosome imbalance was detected in 9 of 10 polar bodies. A variety of chromosomes were aneuploid, but chromosomal size was found to be an important predisposing factor. In three cases, the resulting embryos could be tested using fluorescence in situ hybridization, and in each case the CGH diagnosis was confirmed. A single embryo could be recommended for transfer on the basis of the CGH data, but no pregnancy ensued. Conclusion(s): Evidence suggests that preferential transfer of chromosomally normal embryos can improve IVF outcomes. However, current PGD protocols do not allow analysis of every chromosome, and therefore a proportion of abnormal embryos remains undetected. We describe a method that allows every chromosome to be assessed in polar bodies and oocytes. The technique was accurate and allowed identification of aneuploid embryos that would have been diagnosed as normal by standard PGD techniques. As well as comprehensive cytogenetic analysis, this protocol permits simultaneous testing for multiple single-gene disorders.

Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays

OBJECTIVE To develop and validate a whole genome amplification and single nucleotide polymorphism (SNP) microarray protocol for accurate single cell 24 chromosome aneuploidy screening. DESIGN Prospective, randomized, and blinded study. SETTING Academic reproductive medicine center. PATIENT(S) Multiple euploid and aneuploid cell lines were obtained from a public repository and blastomeres were obtained after biopsy of cleavage stage embryos from 78 patients undergoing IVF. MAIN OUTCOME MEASURE(S) Accuracy of copy number assignment and consistency of individual SNPs, whole chromosomes, and single cell aneuploidy status were determined. INTERVENTION(S) None. RESULT(S) Single cells extracted from karyotypically defined cell lines provided 99.2% accuracy for individual SNPs, 99.8% accuracy for whole chromosomes, and 98.6% accuracy when applying a quality control threshold for the overall assignment of aneuploidy status. The concurrence for more than 80 million SNPs in 335 single blastomeres was 96.5%. CONCLUSION(S) We have established and validated a SNP microarray-based single cell aneuploidy screening technology. Clinical validation studies are underway to determine the predictive value of this methodology.

Incidence and spectrum of chromosome abnormalities in spontaneous abortions: New insights from a 12-year study

Purpose: Despite advances in harvesting and culturing techniques, analysis of the impact of these improvements on the observed frequency of chromosomal abnormalities in spontaneous abortions (SAB) has not been determined. We sought to evaluate the effect of these refinements on the success rate of our cultures and on the resulting frequency of detected chromosomal abnormalities.Methods: Between 1990 and 2002, 2301 specimens obtained from the products of conception (POC) of SABs were submitted to our laboratory for cytogenetic analysis. Due to refinements in specimen processing and culture techniques introduced at the end of 1997, our data were analyzed for two periods: Period A from 1990 through 1997 with 907 eligible specimens and Period B from 1998 through 2002 with 1273 eligible specimens.Results: Modifications in physician communication and sample processing contributed to significant improvements in the culture success rate and in the ratio of male-to-female cases with normal karyotypes. Additionally, increased detection of trisomic, triploid, and multiple aneuploid cases in Period B resulted in a significant increase in the percentage of cases with abnormal karyotypes (42.8% in Period A vs. 65.8% in Period B). Monosomy X accounted for < 10% of all abnormalities in Period B. Eighty five multiple aneuploid karyotypes, including 57 double trisomies, comprised 7.7% of our 1099 abnormal cases. These karyotypes were detected predominantly in POCs from the older women in our study. This collection of multiple aneuploidies is the largest published to date and includes abnormalities not reported in prior studies. We also present a table empirically derived from the data in Period B that indicates the likelihood of a specific abnormal karyotype based on maternal age. The table can be utilized by health care providers, who counsel patients after a spontaneous miscarriage.Conclusion: Improvements in laboratory technique have led to reduced contamination and growth failure of POCs, irrespective of maternal age. This in turn has led to a more balanced male-to-female ratio and to the detection of an increased number of abnormal cases.

Deregulated Overexpression of hCdt1 and hCdc6 Promotes Malignant Behavior

The accurate execution of DNA replication requires a strict control of the replication licensing factors hCdt1 and hCdc6. The role of these key replication molecules in carcinogenesis has not been clarified. To examine how early during cancer development deregulation of these factors occurs, we investigated their status in epithelial lesions covering progressive stages of hyperplasia, dysplasia, and full malignancy, mostly from the same patients. Abnormal accumulation of both proteins occurred early from the stage of dysplasia. A frequent cause of unregulated hCdc6 and hCdt1 expression was gene amplification, suggesting that these components can play a role per se in cancer development. Overexpression of hCdt1 and hCdc6 promoted rereplication and generated a DNA damage response, which activated the antitumor barriers of senescence and apoptosis. Generating an inducible hCdt1 cellular system, we observed that continuous stimulus by deregulated hCdt1 led to abrogation of the antitumor barriers and resulted in the selection of clones with more aggressive properties. In addition, stable expression of hCdc6 and hCdt1 in premalignant papilloma cells led to transformation of the cells that produced tumors upon injection into nude mice depicting the oncogenic potential of their deregulation.

SNP microarray-based 24 chromosome aneuploidy screening is significantly more consistent than FISH

Many studies estimate that chromosomal mosaicism within the cleavage-stage human embryo is high. However, comparison of two unique methods of aneuploidy screening of blastomeres within the same embryo has not been conducted and may indicate whether mosaicism has been overestimated due to technical inconsistency rather than the biological phenomena. The present study investigates the prevalence of chromosomal abnormality and mosaicism found with two different single cell aneuploidy screening techniques. Thirteen arrested cleavage-stage embryos were studied. Each was biopsied into individual cells (n = 160). The cells from each embryo were randomized into two groups. Those destined for FISH-based aneuploidy screening (n = 75) were fixed, one cell per slide. Cells for SNP microarray-based aneuploidy screening (n = 85) were put into individual tubes. Microarray was significantly more reliable (96%) than FISH (83%) for providing an interpretable result (P = 0.004). Markedly different results were obtained when comparing microarray and FISH results from individual embryos. Mosaicism was significantly less commonly observed by microarray (31%) than by FISH (100%) (P = 0.0005). Although FISH evaluated fewer chromosomes per cell and fewer cells per embryo, FISH still displayed significantly more unique genetic diagnoses per embryo (3.2 ± 0.2) than microarray (1.3 ± 0.2) (P < 0.0001). This is the first prospective, randomized, blinded and paired comparison between microarray and FISH-based aneuploidy screening. SNP microarray-based 24 chromosome aneuploidy screening provides more complete and consistent results than FISH. These results also suggest that FISH technology may overestimate the contribution of mitotic error to the origin of aneuploidy at the cleavage stage of human embryogenesis.

Oncogene-induced replication stress preferentially targets common fragile sites in preneoplastic lesions. A genome-wide study

Common fragile sites (CFSs) are regions of the genome prone to breakage by replication inhibitors (extrinsic replication stress). Recently, we and others observed that oncogene-induced replication stress (RS) induces DNA damage from the earliest stages of cancer. Our aim was to perform a genome-wide analysis in precancerous and cancerous experimental models to examine whether allelic imbalance occurs within CFSs. Subsequently, CFSs sequence characteristics were assessed. We used a growth-factor-induced human skin hyperplasia and a H-ras-induced mouse hyperplastic urothelium as preneoplastic models, along with an inducible U2OS-CDT1Tet-ON cancer cell line model, all bearing established oncogene-induced RS stimuli. Human DNA was analysed with Affymetrix SNP microarrays, while mouse DNA was analysed with Nimblegen array CGH. We studied 56 aphidicolin-type CFSs and 1914 regions of control, nonfragile DNA. Our theoretical in silico analysis spanned 2.16 billion nonoverlapping bases on human chromosomes 1–22. Our results provide direct experimental evidence indicating that genomic alterations were more common within CFSs in epidermal and urothelial preneoplastic lesions as well as in cancer. CFSs were on average less flexible than nonfragile regions, contained more guanine–cytosine (GC) and Alu sequences. Importantly, regions with loss-of-heterozygosity were also less flexible and had a higher Alu percentage.

Karyotype versus Microarray Testing for Genetic Abnormalities after Stillbirth

BACKGROUND Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true prevalence may be higher. Unlike karyotype analysis, microarray analysis does not require live cells, and it detects small deletions and duplications called copy-number variants. METHODS The Stillbirth Collaborative Research Network conducted a population-based study of stillbirth in five geographic catchment areas. Standardized postmortem examinations and karyotype analyses were performed. A single-nucleotide polymorphism array was used to detect copy-number variants of at least 500 kb in placental or fetal tissue. Variants that were not identified in any of three databases of apparently unaffected persons were then classified into three groups: probably benign, clinical significance unknown, or pathogenic. We compared the results of karyotype and microarray analyses of samples obtained after delivery. RESULTS In our analysis of samples from 532 stillbirths, microarray analysis yielded results more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided better detection of genetic abnormalities (aneuploidy or pathogenic copy-number variants, 8.3% vs. 5.8%; P=0.007). Microarray analysis also identified more genetic abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P=0.02) and 67 stillbirths with congenital anomalies (29.9% vs. 19.4%, P=0.008). As compared with karyotype analysis, microarray analysis provided a relative increase in the diagnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum stillbirths, and 53.8% in stillbirths with anomalies. CONCLUSIONS Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.).