Correlating genomic copy number alterations with clinicopathologic findings in 75 cases of hepatocellular carcinoma

Background Oligonucleotide array comparative genomic hybridization (aCGH) analysis has been used for detecting somatic copy number alterations (CNAs) in various types of tumors. This study aimed to assess the clinical utility of aCGH for cases of hepatocellular carcinoma (HCC) and to evaluate the correlation between CNAs and clinicopathologic findings. Methods aCGH was performed on 75 HCC cases with paired DNA samples from tumor and adjacent nontumor tissues. Survival outcomes from these cases were analyzed based on Barcelona-Clinic Liver Cancer Stage (BCLC), Edmondson-Steiner grade (E-S), and recurrence status. Correlation of CNAs with clinicopathologic findings was analyzed by Wilcoxon rank test and clustering vs. K means. Results The survival outcomes indicated that BCLC stages and recurrence status could be predictors and E-S grades could be a modifier for HCC. The most common CNAs involved gains of 1q and 8q and a loss of 16q (50%), losses of 4q and 17p and a gain of 5p (40%), and losses of 8p and 13q (30%). Analyses of genomic profiles and clusters identified that losses of 4q13.2q35.2 and 10q22.3q26.13 seen in cases of stage A, grade III and nonrecurrence were likely correlated with good survival, while loss of 1p36.31p22.1 and gains of 2q11.2q21.2 and 20p13p11.1 seen in cases of stage C, grade III and recurrence were possibly correlated with worst prognosis. Conclusions These results indicated that aCGH analysis could be used to detect recurrent CNAs and involved key genes and pathways in patients with HCC. Further analysis on a large case series to validate the correlation of CNAs with clinicopathologic findings of HCC could provide information to interpret CNAs and predict prognosis.

Correlating genomic copy number alterations with clinicopathologic findings in a case series of hepatocellular carcinoma

Background: Oligonucleotide array comparative genomic hybridization (aCGH) analysis has been used for detecting somatic copy number alterations (CNAs) in various types of tumors. This study aimed to assess the clinical utility of aCGH for a case series of hepatocellular carcinoma (HCC) and to evaluate the correlation between CNAs and clinicopathologic findings.Methods: Survival outcomes from this case series were analyzed based on Barcelona-Clinic Liver Cancer Stage (BCLC), Edmondson-Steiner grade (E-S), and recurrence status. aCGH was performed on 75 HCC cases with paired DNA samples from tumor and adjacent nontumor tissues. Correlation of CNAs with clinicopathologic findings was analyzed by Wilcoxon rank test and clustering vs. K means.Results: The survival outcomes indicated that BCLC stages and recurrence status could be predictors and E-S grades could be a modifier for HCC. The most common CNAs involved gains of 1q and 8q and a loss of 16q (50%), losses of 4q and 17p and a gain of 5p (40%), and losses of 8p and 13q (30%). Analyses of genomic profiles and clusters identified that losses of 4q13.2q35.2 and 10q22.3q26.13 seen in cases of stage A, grade III and nonrecurrence were likely correlated with good survival, while loss of 1p36.31p22.1 and gains of 2q11.2q21.2 and 20p13p11.1 seen in cases of stage C, grade III and recurrence were possibly correlated with worst prognosis. Conclusions: These results indicated that aCGH analysis could be used to detect recurrent CNAs and involved key genes and pathways in patients with HCC. Further analysis on a large case series to validate the correlation of CNAs with clinicopathologic findings of HCC could provide information to interpret CNAs and predict prognosis.

Evaluation of extracellular vesicles and gDNA from culture medium as a possible indicator of developmental competence in human embryos

Summary Human embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.

Correlating Genomic Copy Number Alterations with Clinicopathologic Findings in A Case Series of Hepatocellular Carcinoma

Background: Oligonucleotide array comparative genomic hybridization (aCGH) analysis has been used for detecting somatic copy number alterations (CNAs) in various types of tumors. This study aimed to assess the clinical utility of aCGH for a case series of hepatocellular carcinoma (HCC) and to evaluate the correlation between CNAs and clinicopathologic findings.Methods: Survival outcomes from this case series were analyzed based on Barcelona-Clinic Liver Cancer Stage (BCLC), Edmondson-Steiner grade (E-S), and recurrence status. aCGH was performed on 75 HCC cases with paired DNA samples from tumor and adjacent nontumor tissues. Correlation of CNAs with clinicopathologic findings was analyzed by Wilcoxon rank test and clustering vs. K means. Results: The survival outcomes indicated that BCLC stages and recurrence status could be predictors and E-S grades could be a modifier for HCC. The most common CNAs involved gains of 1q and 8q and a loss of 16q (50%), losses of 4q and 17p and a gain of 5p (40%), and losses of 8p and 13q (30%). Correlation and clustering analyses noted that losses of 4q13.2q35.2 and 10q22.3q26.13 seen in cases of stage A, grade III and nonrecurrence were likely associated with good survival, while loss of 1p36.31p22.1 and gains of 2q11.2q21.2 and 20p13p11.1 seen in cases of stage C, grade III and recurrence were possibly associated with worst prognosis. Conclusions: These results indicated that aCGH analysis could be used to detect recurrent CNAs and involved key genes and pathways in patients with HCC. Further analysis on a large case series to validate the association of CNAs with clinicopathologic findings of HCC could provide information to interpret CNAs and predict prognosis.

Familial Infertility (Azoospermia and Cryptozoospermia) in Two Brothers—Carriers of t(1;7) Complex Chromosomal Rearrangement (CCR): Molecular Cytogenetic Analysis

Structural aberrations involving more than two breakpoints on two or more chromosomes are known as complex chromosomal rearrangements (CCRs). They can reduce fertility through gametogenesis arrest developed due to disrupted chromosomal pairing in the pachytene stage. We present a familial case of two infertile brothers (with azoospermia and cryptozoospermia) and their mother, carriers of an exceptional type of CCR involving chromosomes 1 and 7 and three breakpoints. The aim was to identify whether meiotic disruption was caused by CCR and/or genomic mutations. Additionally, we performed a literature survey for male CCR carriers with reproductive failures. The characterization of the CCR chromosomes and potential genomic aberrations was performed using: G-banding using trypsin and Giemsa staining (GTG banding), fluorescent in situ hybridization (FISH) (including multicolor FISH (mFISH) and bacterial artificial chromosome (BAC)-FISH), and genome-wide array comparative genomic hybridization (aCGH). The CCR description was established as: der(1)(1qter->1q42.3::1p21->1q42.3::7p14.3->7pter), der(7)(1pter->1p2 1::7p14.3->7qter). aCGH revealed three rare genes variants: ASMT, GARNL3, and SESTD1, which were ruled out due to unlikely biological functions. The aCGH analysis of three breakpoint CCR regions did not reveal copy number variations (CNVs) with biologically plausible genes. Synaptonemal complex evaluation (brother-1; spermatocytes II/oligobiopsy; the silver staining technique) showed incomplete conjugation of the chromosomes. Associations between CCR and the sex chromosomes (by FISH) were not found. A meiotic segregation pattern (brother-2; ejaculated spermatozoa; FISH) revealed 29.21% genetically normal/balanced spermatozoa. The aCGH analysis could not detect smaller intergenic CNVs of few kb or smaller (indels of single exons or few nucleotides). Since chromosomal aberrations frequently do not affect the phenotype of the carrier, in contrast to the negative influence on spermatogenesis, there is an obvious need for genomic sequencing to investigate the point mutations that may be responsible for the differences between the azoospermic and cryptozoospermic phenotypes observed in a family. Progeny from the same parents provide a unique opportunity to discover a novel genomic background of male infertility.

80 Evaluation of extracellular vesicles from culture medium of human embryos as a possible method of pre-implantation genetic diagnosis

Noninvasive methods are the clue to increase the efficiency of invitro-derived embryo selection without decreasing their competence. Embryos selection based on their morphology is the most used method but only 40% of selected embryos are able to implant and develop correctly. In humans, pre-implantation genetic diagnosis increases the efficiency of selection by excluding embryos with chromosomal abnormalities. However, pre-implantation genetic diagnosis needs embryonic cells, which might compromise embryo viability. On the other hand, embryos release extracellular vesicles (EVs: microvesicles and exosomes) to the culture medium that contain biological cargo-like proteins and mRNA lipids, and might contain genomic DNA (gDNA). For this study we evaluated the culture medium from embryos generated by intracytoplasmic sperm injection in a certified fertility clinic. Embryos were cultured in Global Total serum-free medium. The embryos were assessed at Day 3 of development and classified in three categories: top, fair, and poor quality. Corresponding medium was collected for isolation of EVs. The nature of EVs was confirmed by their size and concentration using nanoparticle tracking analysis (NTA), presence of surface markers (CD9, CD63, CD81, and CD40L), and morphology using transmission electron microscopy. A correlation analysis between NTA results (EV size and concentration) and embryo quality was performed. To evaluate chromosomal abnormalities of gDNA present in isolated EVs from embryo culture medium, microarray-based comparative genomic hybridization (aCGH) assay was performed. In a second experiment, aCGH analysis was performed and compared between arrested embryos and EVs isolated from corresponding culture medium. Isolated nanoparticles from embryo culture medium were positive to all markers CD9 (30.9%), CD63 (27.2%), CD81 (31.7%), CD40L (8.7%) and had a morphology accordingly to exosomes. The analysis of NTA data indicated that top-quality embryos had EVs with higher diameter (mean: 112.17nm, mode: 91.74nm) than embryos classified as fair (mean: 108.02nm, mode: 89.67nm) and poor quality (mean: 102.78nm, mode: 88.17 nm; P&lt;0.05). The aCGH analysis showed the representation of the 23 pairs of chromosomes in EVs from culture medium and the chromosomal abnormalities were detected in chromosome 4 (C4: 6/15 (40%)) and chromosome 13 (C13: 6/15 (40%)). In the second experiment, the aCGH assay also showed abnormalities in different chromosomes from samples of EVs from culture medium (24.9%) and were more frequent than those observed in the arrested embryos (8.7%; P=0.03). However, the rate of similitude in chromosomal abnormalities between EVs and their respective embryo was 70-80%. In conclusion, the size and gDNA of EVs from culture medium might be an alternative to evaluate the competence of human embryos. This research was supported by FONDECYT-1170310 and Corfo 17Cote-72437, Chile.

Genomic deletions upstream of lamin B1 lead to atypical autosomal dominant leukodystrophy

ObjectiveClinical, radiologic, and molecular analysis of patients with genomic deletions upstream of the LMNB1 gene.MethodsDetailed neurologic, MRI examinations, custom array comparative genomic hybridization (aCGH) analysis, and expression analysis were performed in patients at different clinical centers. All procedures were approved by institutional review boards of the respective institutions.ResultsFive patients from 3 independent families presented at ages ranging from 32 to 52 years with neurologic symptoms that included progressive hypophonia, upper and lower limb weakness and spasticity, and cerebellar dysfunction and MRIs characterized by widespread white matter alterations. Patients had unique nonrecurrent deletions upstream of the LMNB1, varying in size from 250 kb to 670 kb. Deletion junctions were embedded in repetitive elements. Expression analysis revealed increased LMNB1 expression in patient cells.ConclusionsOur findings confirmed the association between LMNB1 upstream deletions and leukodystrophy previously reported in a single family, expanding the phenotypic and molecular description of this condition. Although clinical and radiologic features overlapped with those of autosomal dominant leukodystrophy because of LMNB1 duplications, patients with deletions upstream of LMNB1 had an earlier age at symptom onset, lacked early dysautonomia, and appeared to have lesser involvement of the cerebellum and sparing of the spinal cord diameter on MRI. aCGH analysis defined a smaller minimal critical region required for disease causation and revealed that deletions occur at repetitive DNA genomic elements. Search for LMNB1 structural variants (duplications and upstream deletions) should be an integral part of the investigation of patients with autosomal dominant adult-onset leukodystrophy.

The Genomic Landscape of PAX5, IKZF1, and CDKN2A/B Alterations in B-Cell Precursor Acute Lymphoblastic Leukemia

We present a comprehensive comparison of PAX5,IKZF1, and CDKN2A/B abnormalities in 21 B-cell precursor acute lymphoblastic leukemia (B-ALL) patients studied by aCGH and gene-specific FISH assays. In our cohort of B-ALL patients, alterations of IKZF1, PAX5, and CDKN2A/B were detected by aCGH analysis in 43, 52, and 57% of samples, respectively. Deletions of IKZF1 were present in 9 samples, including 5 cases positive for both PAX5 and IKZF1 deletions, implying digenic impairment. Furthermore, all cases with IKZF1 deletions also had additional genomic alterations, including BCR-ABL1 gene fusions, PAX5 deletions, CDKN2A/B deletions, and FLT3 amplification. Deletions of CDKN2A/B represented the most frequent abnormalities in our group of patients. Our study demonstrates the high incidence of PAX5, IKZF1, and CDKN2A/B alterations in B-ALL detected by aCGH analysis. Due to the small size and variability in the deletion breakpoints, FISH studies showed false-negative results in 10, 40, and 28% of the samples tested for the IKZF1,PAX5, and CDKN2A/B gene deletions, respectively. The PAX5 and IKZF1 abnormalities are highly specific to B-ALL and can be used as diagnostic markers. Moreover, IKZF1 alterations frequently coexist with a BCR-ABL gene fusion. Our study revealed multiple additional B-ALL-specific genomic alterations and showed that aCGH is a more sensitive method than FISH, allowing whole genome profiling and identification of aberrations of diagnostic and prognostic significance in patients with B-ALL.