Search

Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Aims The existence of malignant mesothelioma in situ (MIS) is often postulated, but there are no accepted morphological criteria for making such a diagnosis. Methods and results Here we report two cases that appear to be true MIS on the basis of in-situ genomic analysis. In one case the patient had repeated unexplained pleural unilateral effusions. Two thoracoscopies 9 months apart revealed only visually normal pleura. Biopsies from both thoracoscopies showed only a single layer of mildly reactive mesothelial cells. However, these cells had lost BRCA1-associated protein 1 (BAP1) and showed loss of cyclin-dependent kinase inhibitor 2 (CDKN2A) (p16) by fluorescence in-situ hybridisation (FISH). NF2 was not deleted by FISH but 28% of the mesothelial cells showed hyperploidy. Six months after the second biopsy the patient has persisting effusions but no evidence of pleural malignancy on imaging. The second patient presented with ascites and minimal omental thickening on imaging, but no visual evidence of tumour at laparoscopy. Omental biopsy showed a single layer of minimally atypical mesothelial cells with rare tiny foci of superficial invasion of fat. BAP1 immunostain showed loss of nuclear BAP1 in all the surface mesothelial cells and the invasive cells. There was CDKN2A deletion, but no deletion of NF2 by FISH. Conclusions These cases show that morphologically bland single-layered surface mesothelial proliferations with molecular alterations seen previously only in invasive malignant mesotheliomas exist, and presumably represent malignant MIS. More cases are need to understand the frequency of such changes and the time-course over which invasive tumour develops.

Cancer cells are subjected to constant selection by the immune system, meaning that tumors that become clinically manifest have managed to subvert or hide from immunosurveillance. Immune control can be facilitated by induction of autophagy, as well as by polyploidization of cancer cells. While autophagy causes the release of ATP, a chemotactic signal for myeloid cells, polyploidization can trigger endoplasmic reticulum stress with consequent exposure of the "eat-me" signal calreticulin on the cell surface, thereby facilitating the transfer of tumor antigens into dendritic cells. Hence, both autophagy and polyploidization cause the emission of adjuvant signals that ultimately elicit immune control by CD8+ T lymphocytes. We investigated the possibility that autophagy and polyploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. Mass spectrometry led to the identification of peptides that were presented on major histocompatibility complex (MHC) class I molecules in an autophagy-dependent fashion or that were specifically exposed on the surface of polyploid cells, yet lost upon passage of such cells through immunocompetent (but not immunodeficient) mice. However, the preferential recognition of autophagy-competent and polyploid cells by the innate and cellular immune systems did not correlate with the preferential recognition of such peptides in vivo. Moreover, vaccination with such peptides was unable to elicit tumor growth-inhibitory responses in vivo. We conclude that autophagy and polyploidy increase the immunogenicity of cancer cells mostly by affecting their adjuvanticity rather than their antigenicity.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. It is characterized by highly complex karyotypes with structural and numerical chromosomal alterations. The observed OS-specific characteristics in localization and frequencies of chromosomal breakages strongly implicate a specific set of responsible driver genes or a specific mechanism of fragility induction. In this study, a comprehensive assessment of somatic copy number alterations (SCNAs) was performed in 160 OS samples using whole-genome CytoScan High Density arrays (Affymetrix, Santa Clara, CA). Genes or regions frequently targeted by SCNAs were identified. Breakage analysis revealed OS specific unstable regions in which well-known OS tumor suppressor genes, including TP53, RB1, WWOX, DLG2 and LSAMP are located. Certain genomic features, such as transposable elements and non-B DNA-forming motifs were found to be significantly enriched in the vicinity of chromosomal breakage sites. A complex breakage pattern-chromothripsis-has been suggested as a widespread phenomenon in OS. It was further demonstrated that hyperploidy and in particular chromothripsis were strongly correlated with OS patient clinical outcome. The revealed OS-specific fragility pattern provides novel clues for understanding the biology of OS.

The aneuploidy frequencies have been stuided in vitro in lymphocytes from couples with recurent abortions, from parents of a trisomic child and from control couples with at least two normal children; c-heterochromatin variants have been analysed on the same samples by length measurements. A significant increase of hyeperploid cells has been observed in the lymphocytes from parents of a trisomic and couples with recurrent abortions as well. However, no consistent correlation has been found so far between c-heterochromatin varients and an increase of aneuploidy.

Background : Antimicrotubule drugs (AMDs), suchas taxol and vincristine, are the most importantaddition to the chemotherapeutic armamentariumagainst human cancers. It has been shown that pro-longed AMD treatment induces hyperploidy inG1-checkpoint-defective cancer cells and that thesehyperploid cells subsequently undergo apoptosis.However, a fraction of these hyperploid cells are ableto survive the prolonged mitotic stress and resumecell-cycle progression.Results: We established hyperploid clones that escapedfrom cell death after AMD treatment from two gliomacell lines, U251MG and U87MG. Subtractive compar-ative genomic hybridization (CGH) analysis revealedthat clones derived from U87MG mainly had chromo-some number changes, but that those from U251MGshowed both numerical and structural chromosomalchanges. Furthermore, numerous aberrations identifiedin U251MG clones were remarkably chromosome-specific, which may have been due to clonal selectionfor cells that have an advantage in growth and/or sur-vival. All clones derived from both cell lines had abnorm-alities in chromosome segregation, and karyotypesof clones were more heterogeneous than those ofparental cells, suggesting that cells having a higherchromosome number are subject to asymmetric chro-mosome segregation, resulting in a heterogeneouskaryotype. All clones derived from U87MG andU251MG increased both centric and acentromericmicronuclei, suggesting the presence of chromosomestructural abnormality.Conclusions: AMD treatment induces hyperploid for-mation and chromosome instability in checkpoint-deficient cancer cells.

We describe a 58-year-old woman with anaplastic multiple myeloma and multiple chromosomal abnormalities. Her karyotype showed extreme hyperploidy with 77 chromosomes. Some of the aberrations were typical of multiple myeloma (+3, +5, +15, +19, +21, t(11; 14)(q13; q32)), others were characteristic of the aggressive anaplastic myeloma (+8), t(11; 14)(q13; q32), while three chromosomal abnormalities (t(11; 20)(p11; q13); t(4; 7)(q31; q11); and t(14; 20)(q24; q13)) have not been, to the best of our knowledge, described previously in the literature. The fulminant course of the disease confirms the poor prognosis of multiple karyotypic abnormalities in myeloma.

Fluorescent in situ hybridization (FISH) with alpha satellite DNA probes for chromosomes 11 and X were applied to normal, atypical, and dysplastic cervical-vaginal cytology smears to evaluate the detection of hyperploidy in suspected abnormal cells. Forty-six cases were obtained from fixed archival material. Eight cases with a morphological diagnosis of within normal limits (WNL) were directly selected to use as controls. The other 38 cases were blinded as study cases. These included five WNL, six ASCUS, six SIL-LG, 16 SIL-HG, four invasive squamous cell carcinomas, and one case of adenocarcinoma of the cervix. Cells with chromosome copy numbers suggesting hyperploidy (3-4 signals per chromosome specific probe) were found more often in higher grade dysplasia (Bethesda class SIL-HG) cases and less often in lower grade lesions (SIL-LG). All cases morphologically diagnosed as WNL were found to have normal copy number except for one control case which was hyperploid and, upon reexamination of the original slides, was upgraded from normal to atypical squamous cells of undetermined significance (ASCUS). Our FISH results are similar to those of previous studies involving flow cytometry and morphometric cytometry in which changes in ploidy correlated with progression toward higher grade lesions. However, FISH with enumeration probes offers a higher resolution view of the genome than is possible with flow cytometry or morphometry by allowing detection of specific chromosome changes in small numbers of affected cells in a routine cervical smear, and it may have the capacity to detect those cases in which progression toward high grade dysplasias is more likely.

Chromosome segregation at meiosis I was studied in oocytes and spermatocytes of four different Robertsonian (Rb) heterozygous mouse stocks by cytogenetic analysis of meiotic products. Two Rb heterozygotes spontaneously yielded high frequencies of unbalanced oocytes. In one case, Rb(2.18)Rma, the excess hyperploidy was mainly accounted for by nondisjunction of normal bivalents, suggesting a generalized impairment of meiotic segregation. In each stock, frequencies of hyperploid spermatocytes were either not significantly different or significantly lower than the corresponding frequencies in the oocytes. This confirmed the greater risk of segregational errors in female than in male carriers of the same Rb metacentric. The hypothesis that an error prone system of meiotic segregation, such as the trivalent configuration of single Rb heterozygous oocytes, could be hypersensitive to chemically induced malsegregation was tested by injecting Rb heterozygous females with low doses of vinblastine (VBL). An intraperitoneal injection of 0.06 or 0.09 mg/kg VBL before the first meiotic division significantly increased the spontaneous frequency of hyperploid oocytes, inducing segregational errors of both the trivalent and normal bivalents. The comparison of these data with VBL effects in B6C3F1 mice showed that single Rb heterozygous oocytes are more sensitive to VBL-induced meiotic aneuploidy than oocytes with a standard karyotype. Although segregation distortion has been repeatedly shown in the progeny of Rb heterozygous mice with a significant excess of all telocentric balanced offspring, it has never been demonstrated whether this is a primary event occurring during meiotic segregation or a consequence of selective postconceptional death. In this study, we showed that preferential segregation occurred during female meiosis in all the Rb stocks tested. When segregation distortion was analyzed separately in balanced and unbalanced oocytes, the latter did not show preferential segregation, suggesting that, when the two telocentrics segregated from each other, then the metacentric was randomly directed to the ovum or the polar body.

A procedure to enhance the concurrent yield of large numbers of spermatogonial, meiotic I, and meiotic II metaphases from Chinese hamster testes has been developed. Animals were injected intraperitoneally with 5, 10, 20, or 40 mg of colchicine/kg and killed 3 hr after the treatment. Both testes from each animal were excised; one testis was minced, and germ cells were isolated by using 0.1% trypsin solution; the other testis was minced, and germ cells were isolated by using a conventional procedure. Chromosomal preparations were made with a standard air-drying technique. Examination of slides revealed that with the trypsin-isolation procedure the concurrent yields of spermatogonial, meiotic I, and meiotic II metaphases were significantly higher (P less than .05) than the yields obtained with the conventional procedure. Furthermore, 40 mg of colchicine/kg produced maximum numbers of all three types of germ cell metaphases. At this maximum concentration, metaphases were stable with no evidence of structural aberrations, and the frequency of hyperploidy was not significantly different (P greater than .05) from hypoploidy.

Numerical chromosomal aberrations (aneuploidy) are a major factor in pregnancy wastage, birth defects, and mental retardation. Consequently, effective cytogenetic procedures in mammalian gamates are required for studying mechanisms and causes of chromosomal nondisjunction. Our aims were to determine for oocytes from superovulating prepubertal mice: (1) the yield of chromosomally scorable ova, following application of a double fixation procedure: (2) the background level of aneuploidy, and (3) the sensitivity to induction of aneuploidy (by methotrexate). Superovulation was induced in C129F1 hybrid mice, 22-24 days old, with pregnant mare serum and human chorionic gonadotropin (HCG). Diurnal photoperiodicity and injections were scheduled to assure HCG-induced ovulation of known timing. Methotrexate (200 mg/kg) was given at 3 hr and ova were recovered at 15 hr after HCG. We describe the adaptation of a double fixation procedure to mouse oocytes. Methotrexate led to significantly increased hypoploidy (2 1/2-fold) but not to the hyperploidy reported by others for adult mice. There was a high yield of ova with exactly countable chromosomes (average of 9.5 ova per mouse). Concomitantly, the background level of aneuploidy was very low (0/465 scorable ova were hyperploid). Given the additional advantages of economy and convenience, the superovulating 3-week-old mouse could be an effective source of ova for testing environmental agents for their aneuploidy-inducing potential; however, further studies are needed to establish the degree to which such ova are susceptible to aneuploidy induction.

Flow cytometry studies of the DNA distribution of tumor cells from 92 human breast cancers showed measurable aneuploidy (hyperploidy) in 83 cases (92%). The DNA ploidy values were unimodal in each case, but there was a bimodal distribution for the entire series. One group of tumors had a diploid or near diploid DNA distribution and a second group had ploidy levels from triploid to tetraploid or higher. The tumors with lower DNA ploidy (at or near diploid) tended to be histologically low grade, cytologically more orderly and estrogen-binding positive; those with higher DNA ploidy were more likely to be higher grade, more anaplastic, and estrogen-binding negative.

The chromosome patterns of 17 adenomas and 7 adenocarcinomas of the colon were studied using a direct technique. The typical adenomas showed definite abnormalities of chromosome pattern. Many pseudodiploids and many cells with counts of 47 were identified. These latter appeared due to a trisomy. Nondisjunction with persistence of the resulting abnormal stemline is postulated. Those adenomas with areas of histologic atypia showed greater hyperploidy and more numerous individually abnormal chromosomes. Hyperploidy was also marked in three adenomas which were in whole or part villous. These two groups were similar to the markedly hyperploid and aneuploid carcinomas studied. Two poorly differentiated adenocarcinomas were much less hyperploid than the remaining five better differentiated adenocarcinomas.

It appears that the chance of an in vitro fertilization (IVF) embryo to produce a pregnancy carried to term is about 10%.1 Consequently, one of the most important factors in increasing the pregnancy rate after in vitro fertilization and embryo transfer is replacement of multiple embryos.2 When we consider that the estimated human fecundity rate is about 25%,3 it seems likely that the quality of the embryo is one determining factor for a successful pregnancy rate. Different types of defects of gametes and embryos might contribute to the explanation of why the vast majority of IVF embryos do not give rise to any sign of pregnancy after replacement. It is well documented that chromosomal abnormalities among first trimester spontaneous abortions occur at a rate of about 60%, indicating that chromosomal abnormalities might be common in human gametes and early development stages.4 Abnormal karyotypes found were aneuploidy (i.e., complements with one extra or one missing chromosome) and polyploidy. Recently, a new technique allowing direct analysis of the chromosomal constitution of human spermatozoa has been reported.5 This technique utilizes the ability of capacitated human spermatozoa to penetrate zona-free hamster oocytes. Five to 10% of penetrating human spermatozoa have shown chromosomal abnormalities. Various degrees of hyperploidy and hypoploidy ranging from three extra to three missing chromosomes were found in a series of 1000 human spermatozoal chromosome constitutions.6 It is interesting to note that among spontaneous first trimester abortions such serious numerical abnormalities have not been found.4