Abnormal Cell Lines Research Articles

Trisomy 16 and trisomy 16 mosaicism: A review

A review of all prenatal and postnatal diagnoses of trisomy 16 and trisomy 16 mosaicism was carried out in the context of the current understanding of confined placental mosaicism and uniparental disomy (UPD). The prenatal detection of trisomy 16 cells is associated with a high probability of fetal death, preterm delivery, intrauterine growth retardation, and fetal anomalies. Birth defects were typical of those seen in nonmosaic partial duplications of chromosome 16. Surprisingly, anomalies were sometimes limited to a single organ and included some relatively common isolated defects such as a ventricular septal defect, hypospadias, imperforate anus, inguinal hernia, and clubfoot. The risk for abnormality appeared to be higher in those pregnancies in which trisomy 16 cells were identified in amniotic fluid compared to the detection in chorionic villi samples. Contrary to nonmosaic trisomy 16 with an excess of males, mosaic trisomy 16 shows an excess of female karyotypes. Following the prenatal detection of trisomy 16 cells, aneuploid cells are almost never found in fetal or neonatal lymphocytes. Studies on fibroblasts also often fail to confirm the presence of the abnormal cell line even in cases in which multiple anomalies are present. It is likely that trisomy 16 cells are sometimes present in the early developing embryo even though subsequent cytogenetic studies on fetal or neonatal tissues may not detect any aneuploid cells. UPD can be excluded as a mechanism for those anomalies that are common to mosaic trisomy 16 and nonmosaic partial duplications. The term "occult mosaicism" is suggested to describe the situation in which the presence of an abnormal cell line is suspected on the basis of clinical data but unproven by laboratory analysis.

Anterior segment dysgenesis in mosaic Turner syndrome

AIMS/BACKGROUNDFemales with Turner syndrome commonly exhibit ophthalmological abnormalities, although there is little information in the literature documenting findings specific to Turner syndrome mosaics. Ophthalmic findings are described in four patients...

Mosaicism

Mosaicism detected in the cytogenetic analysis of chorionic villi or cultured amniocytes can present a difficult and at times impossible interpretative dilemma. The finding may be indicative of a generalized fetal mosaicism. However, in the majority of cases, the abnormal cell line is representative of either an in-vitro event or a chromosomal error that is restricted to the extra-embryonic tissues. Advances in laboratory medicine have provided insight into the determination of which cases of true mosaicism have the greatest risk of being clinically significant with regard to the fetal genotype and phenotype. Despite the presence of a normal fetal karyotype, certain chromosomes involved in confined placental mosaiciam may increase the risk of poor perinatal outcome or predispose the fetus or neonate to the adverse effects of genetic imprinting owing to the development of uniparental disomy.

Is the 15-in situ clone protocol necessary to detect amniotic fluid mosaicism?

OBJECTIVE: Our purpose was to evaluate the 15-clone analysis for detecting amniotic fluid mosaicism by the in situ method. STUDY DESIGN: A 10-year review was performed of all amniotic fluid mosaicism cases at two institutions using the in situ method exclusively, with sequential clonal analysis to determine the first and second clone in which the abnormal cell line occurred. RESULTS: Of the 28,497 amniotic fluid samples, 73 met criteria for amniotic fluid mosaicism by in situ method (0.26%). There were 54 cases (0.19%) with potential clinical significance (23 autosome and 31 sex chromosome mosaicism), 49 of the 54 cases (89%) were detected in the first six clones, including 22 of 23 involving autosomes and 27 of 31 involving sex chromosomes. In one of the six cases detected after clone 6 ( 46, XX 47, XX , + 21 ) the mosaic cell line was present in 20% of the clones analyzed and was followed by a voluntary termination of the pregnancy. In the other five cases amniotic fluid mosaicism was present in <20% of the clones; these included one case of 46, XX 47, XX + mar (15% amniotic fluid mosaicism, voluntary termination of pregnancy), two cases of 45, X 46, XY (10% to 12% amniotic fluid mosaicism, both normal at birth), and two cases of 45, X 46, XX (8% amniotic fluid mosaicism, lost to follow-up; 12% amniotic fluid mosaicism, voluntary termination of pregnancy). By limiting the analysis to six clones, ∼20% of analysis time could be saved per case, but one autosomal amniotic fluid mosaicism case per 10,000 samples could potentially be missed. CONCLUSION: Reducing the number of clones analyzed by in situ method could result in increased efficiency, decreased costs, and minimal loss of sensitivity.

An accessory marker derived from chromosome 20 and its co‐existence with a mosaic trisomy 20 cell line

We report a 16-month-old boy with delayed psychomotor development, dysmorphic features, and failure to thrive. He had a mosaic karyotype detected prenatally: mos 46,XY/47,XY, +r(20)/47,XY, +20. After birth, the abnormal cell lines were confirmed in a number of tissues. The small ring chromosome was identified using fluorescence in situ hybridization as derived from chromosome 20. We compared our patient with previously reported cases of mosaic trisomy 20 detected prenatally and associated with an abnormal phenotype. In an attempt to characterize an r(20) syndrome, we also compared our case with two similar reports in the literature.

Characterisation of a complex karyotype in AML M4EO using fluorescence in situ hybridisation

Cytogenetic studies of malignant cells can reveal complex karyotypes that cannot be fully described by conventional G-band analysis. Fluorescence in situ hybridisation (FISH) can help to more accurately describe such abnormalities. Acute myelomonocytic leukaemia with eosinophilia (AML M4EO) is associated with karyotypic abnormalities involving rearrangement of chromosome 16q22, most commonly inv(16)(pl3q22). A patient diagnosed as AML M4EO was shown by conventional G-banding to have a complex karyotype with loss of chromosomes 7, 12 and 16. In addition, rearrangements resulting in partial loss of chromosomes 5q and 13q and partial gain of 8q (due to deletion [13] or non- reciprocal translocation with unidentified material [5,8]) and a marker chromosome (of unknown origin) were noted. To investigate the origin of the marker and derivative chromosomes, and in particular to determine if rearrangements involving chromosome 16 were present, FISH studies were performed. Library probes for chromosomes 5, 7, 8, 12 and 16 and a cocktail of 7 centromere and long arm telomere were hybridised to metaphase spreads from cultured bone marrow. FISH showed the presence of several abnormal cell lines. No rearrangement of 16 was found. Chromosome 7 material was found on the marker and on the derivative 8 chromosomes. A combined approach such as this, revealed additional multiple complex rearrangements not detected by routine analysis. In addition, it was useful in delineating minimum regions of chromosome loss. This highlights the value of FISH investigations of chromosomal abnormalities in leukaemias as it has the potential to fully resolve the karyotype.

Direct detection of non-random X chromosome inactivation by use of a transcribed polymorphism in the XIST gene.

As a result of X chromosome inactivation, females are mosaic for cell lineages in which either the paternal or the maternal X chromosome is active, and, if inactivation were random, each lineage should be present at approximately the same frequency. Detection of instances of non-random X inactivation can be important both clinically and for the study of X chromosome inactivation. Identification of a single-base polymorphism in an expressed region of the human XIST gene has permitted the development of a direct PCR-based assay for randomness of X inactivation. Oligonucleotide primers were designed, incorporating the variant base, and conditions established that allowed allele-specific PCR amplification. As the XIST gene is expressed only from the inactive X chromosome, differential amplification of the alleles in cDNA from heterozygotes can be used as an indicator of non-random inactivation. Using this assay, non-random X chromosome inactivation has been demonstrated in chromosomally abnormal cell lines and in lymphocytes from heterozygous, normal females. Virtually complete non-random X inactivation was also shown in a mother and her daughter, suggesting the existence of some familial factor affecting X chromosome inactivation.

Chorionic villi sampling: Laboratory experience with 4,000 consecutive cases

Experience with 4,000 consecutive CVS cases shows that 1) the combination of both the direct and culture methods greatly reduces false diagnoses and maternal cell contamination; 2) the time interval between the sampling procedure and processing of villus specimens influences the quality of direct preparations; 3) maternal cell contamination (MCC) can be minimized with dissection of CVS specimens. We have compiled a large volume of confined placental mosaicism (CPM) cases to serve as a resource in interpreting mosaic cytogenetic findings. It was noted that, in up to 92% of the mosaic cases, the abnormal cell line was confined to the placenta. The frequency of true chromosomal mosaicism was 0.2%, and is not different from that for amniocentesis.

Proliferative verrucous leukoplakia: Four cases with flow cytometric analysis

Proliferative verrucous leukoplakia is a slow-growing but highly aggressive precancerous form of leukoplakia of unknown cause. Proliferative verrucous leukoplakia is thought to possess a continuous spectrum of clinical and histopathologic expression, ranging from simple hyperkeratosis to invasive squamous cell carcinoma. Early diagnosis is difficult because of an initial innocuous character, but multiple and rapid multifocal warty recurrences are common. This article reports four additional archival cases of proliferative verrucous leukoplakia to determine if flow cytometric analysis can be useful in the early diagnosis of proliferative verrucous leukoplakia. Flow cytometric analysis was performed on available formalin-fixed paraffin-embedded specimens (N = 27). Flow cytometric analysis results showed DNA aneuploid cell lines in each proliferative verrucous leukoplakia case studied (DNA index range, 1.1 to 2.6). In all four patients the abnormal cell line DNA index appeared to be maintained throughout the sampling period. The results suggest flow cytometric analysis could be a possible aid in early recognition of proliferative verrucous leukoplakia and might enable aggressive therapy at an earlier stage.

Cytogenetic findings in a new case of adenoid cystic carcinoma arising in sphenoidal sinus

We studied a case of adenoid cystic carcinoma. Cytogenetic analysis was performed on short-term culture, and the karyotype revealed only an abnormal cell line with the following changes: partial trisomy 5q, 6q deletion, monosomy of chromosome 9,der(10)t(10;15), a possible ring chromosome 22, and loss of the Y chromosome. The implication of chromosomes 6 and 9 is considered in relation to the karyotypic evolution of this type of tumor.

Confined placental mosaicism and intrauterine fetal development

Chromosomal mosaicism is the presence of two or more cell lines with different chromosomal complements in an individual derived from a single zygote (which is distinguishable from chimaerism in which an individual is derived from more than one zygote). Mosaicism may involve a number of different karyotypic abnormalities including those for whole sets of chromosomes (polyploidy, including triploidy and tetraploidy), trisomies, sex chromosomal anomalies and chromosomal rearrangements. Mosaicism itself may be present in all the tissues of the body and placenta, or it may only be seen in certain tissues or may be restricted to only the placenta and extraembryonic membranes. The type of mosaicism seen depends upon the stage of development at which it originated, the tissue types to which the abnormal cell lines were distributed and the cell cycle times of both the normal and abnormal cells. The phenotypic effects or consequences of chromosomal mosaicism may be dramatic. During the pregnancy, high levels of mosaicism are seen in early abnormal preimplantation embryos, spontaneous abortions, intrauterine growth restriction and intrauterine fetal death. However, not all cases of mosaicism are serious and the presence of a normal cell line may dilute the effects of an abnormal cell line to such an extent that in many cases mosaicism will remain undetected because the consequences are not severe. Investigations of chromosomal mosaicism were, until recently, restricted to those cells which could be induced to divide and be karyotyped from metaphase analysis. Advances in molecular genetics and cytogenetics have made possible the identification of chromosomal abnormalities, specifically mosaicism in interphase cells. The identification of abnormalities such as confined placental mosaicism and its association with uniparental disomy has led to the recognition that chromosomal mosaicism plays a major role in abnormal human development and dysmorphology (Hall, 1990). An understanding of mosaicism and the role of the placenta during development, including the phenomenon of trisomic zygote rescue, is helping to cast light on problems in prenatal diagnosis, many of which involve confined placental mosaicism.

Acute myelogenous leukemia: implications of acute blast crisis and cardiopulmonary bypass

CUTE MYELOCYTIC leukemia (AML) is a disorder characterized by the expansion of an abnormal white blood cell line. This clonal expansion of cells may infiltrate the bone marrow and other organs, suppressing normal hematopoiesis and inducing dysfunction of the invaded organs. Clinical manifestations of this disease, therefore, result from lack of normal blood cells (primarily due to decreased production) and induced organ dysfunction (secondary to cellular infiltration and/or local hemorrhage). Although the exact incidence of coagulation abnormalities is not known, thrombohemorrhagic complications are certainly common in patients with AML (20% incidence of fatal hemorrhage), especially compared to chronic leukemias; blast crisis accentuates this potential. Although isolated case reports of thrombosis of venous and mammary artery grafts have been reported in patients requiring coronary artery bypass grafting (CABG) with concurrent stable chronic leukemia, nothing has been reported regarding potential thrombohemorrhagic events during acute blast crisis. Hence, a case is presented in which urgent CABG was performed in a patient with AML complicated by a recently diagnosed blast crisis.

Cytogenetic analysis of an adenoid cystic carcinoma of the Bartholin's gland — A rare, semimalignant tumor of the female genitourinary tract.

Abstract Cytogenetic analysis has been performed on short-term cultures from a 56-year-old woman suffering from an adenoid cystic carcinoma of Bartholin's gland. Beside a normal female karyotype, the tumor revealed an abnormal cell line with complex chromosome changes involving the chromosomes 1, 4, 6, 11, 22, and 14. The mainly structural and nonbalanced rearrangements led to the loss of the chromosome segments 1p31→qter, 4q22→q28, 6p12→qter, 11p11.2→pter, 14q24→qter, and 22q13→qter. Clonal numerical aberrations were not observed. To our knowledge, such a tumor has to-date not been cytogenetically investigated.

The use of giant cell tumor conditioned media in cytogenetic studies of hematologic malignancies

The use of conditioned media produced from solid tumor cell lines has been beneficial in the study of hematologic malignancies. Conditioned media from giant cell tumors (GCT), human lung adenocarcinoma, and human bladder carcinoma express growth factors that have been used to stimulate growth of bone marrow cells and improve the quality of the preparations. It has been reported that addition of Lu-CSF1-conditioned media from a lung adenocarcinoma cell line masks abnormalities in cases of acute leukemia [1.] Because we routinely use GCT-CM in bone marrow and leukemic blood cultures for chromosome analysis in our lab, we investigated this potential effect on our case analysis. We have performed a serial study of a 100 cases of hematologic malignancies received for analysis in our lab to determine the effect of the addition of GCT-CM to our culture media with respect to 1) mitotic index, 2) quality of preparation, and 3) differential selection of either chromosomally normal or abnormal cell lines. Our results indicate that the mitotic index and quality of metaphases is enhanced with the addition of GCT media and that there is no difference in the rate of abnormality detection with or without the addition of GCT media.

Cytogenetic analysis of an adenoid cystic carcinoma of the Bartholin's gland: A rare, semimalignant tumor of the female genitourinary tract

Cytogenetic analysis has been performed on short-term cultures from a 56-year-old woman suffering from an adenoid cystic carcinoma of Bartholin's gland. Beside a normal female karyotype, the tumor revealed an abnormal cell line with complex chromosome changes involving the chromosomes 1, 4, 6, 11, 22, and 14. The mainly structural and nonbalanced rearrangements led to the loss of the chromosome segments 1p31→qter, 4q22→q28, 6p12→qter, 11p11.2→pter, 14q24→qter, and 22q13→qter. Clonal numerical aberrations were not observed. To our knowledge, such a tumor has to-date not been cytogenetically investigated.

Survival of fetuses with 45,X: an instructive case and an hypothesis.

The high (greater than 95%) fetal loss rate of 45,X embryos and fetuses has led to the suggestion that fetal survival with this karyotype requires the presence of mosaicism. However, in many instances, even given a "mild Ullrich-Turner syndrome" phenotype, mosaicism is not detected. In a pregnancy studied for advanced maternal age, CVS cultured cells showed 65% 45,X and 35% 46,X,r(X). After termination, 2 fetal tissues showed 95% 45,X cells. It is suggested that infants with the 45,X karyotype likely had mosaicism for a structurally abnormal X or Y chromosome during embryogenesis, but the abnormal cell line disappeared prior to birth.

Nucleotide sequence of prothrombin gene in abnormal prothrombin-producing hepatocellular carcinoma cell lines.

A protein induced by vitamin K absence or antagonist II, PIVKA-II is synthesized in the liver and possesses a structure similar to prothrombin except that ten glutamic acid residues in amino-terminal Gla domain are not completely gamma-carboxylated and are functionally inactive. This protein can be detected in the plasma of patients with hepatocellular carcinoma (HCC) and used as a new tumor marker. To analyze the mechanism of PIVKA-II production in HCC tissue, the prothrombin gene of PIVKA-II-secreting HCC cell lines was sequenced to detect the mutation in the Gla domain and carboxylase recognition site of leader sequence located on exons I and II that may cause the inhibition of carboxylation. Exons I and II and donor and acceptor site of intron I of the prothrombin gene in two HCC cell lines, PLC/PRF/5 and huH-2, were analyzed by polymerase chain reaction (PCR), and the product was sequenced directly. In addition, RNA samples of these cell lines were used for complementary DNA synthesis, followed by PCR and sequencing. The nucleotide sequences of the Gla domain in both HCC cell lines were conserved. One nucleotide change was detected at nt.554 (adenine to guanine), but this did not influence the amino acid sequence. Splicing sites between exons I and II, the leader sequence of the precursor prothrombin, and protease target sites also were conserved as the reported prothrombin gene, and mutations reported for other des-gamma-carboxy coagulation factors were not detected. These results also were confirmed by DNA analysis of seven human fresh-frozen samples (three PIVKA-II-positive HCC samples and four control specimens). The mechanism of PIVKA-II production in HCC is still unclear, but it is not caused by mutation in the prothrombin gene.

Molecular cytogenetic analysis of a medulloblastoma with isochromosome 17 and double minutes

Cytogenetic analysis of a medulloblastoma revealed two abnormal cell lines of 48,XY,+8,+8,−14,+der(14)t(1;14)(q11;p11),i(17q) and 51,XY,+5,+6,+8,+8,−14+20,+der (14)t(1;14)(q11;p11),i(17q),+dmin. The finding of double minute chromosomes in some medulloblastomas has been associated with amplification of the c-myc or N-myc oncogenes. We were unable to detect gene amplification with these probes by Southern blot analysis.

Prenatal diagnosis and outcomes of five cases of mosaicism for an isochromosome of 20q

Five cases of mosaicism for an isochromosome of 20q have been detected from a total of 50,000 cases analysed for prenatal diagnosis by amniocentesis. Karyotypes were designated mos 46,X-/46,X-,i(20q). In all cases, the abnormal cell line was detected in more than one primary culture, thus fulfilling the criterion for true (level III) mosaicism. Indications for prenatal diagnosis were parental anxiety (two cases), low maternal serum alpha-fetoprotein (AFP) (two cases), and high maternal serum AFP (one case). Level II ultrasounds on all five fetuses were normal, and the abnormal cell line was never detected in fetal blood and/or cord blood. All five pregnancies were continued and had normal outcomes, with birth weights ranging from 2.4 to 3.8 kg. The development of all five children has been normal, with the oldest child in the study now 4 years of age. We suggest that the abnormal cell line in each case was of extrafetal origin, and that this may be one of the more common examples of this phenomenon, occurring in approximately 1/10,000 prenatal diagnoses. Mosaicism i(20q) may have been missed in the past because of the higher resolution necessary to detect this subtle change.

Sequential DNA Flow Cytometry in Metastatic Malignant Melanoma

Sequential flow cytometry was performed on 73 metastatic malignant melanomas, derived from 804 primary tumors. Tumor thickness was confirmed an excellent prognostic parameter in primary melanoma, but did not allow reliable predictions in metastatic disease. Also, aneuploidy and genetic heterogeneity, both common in metastatic melanoma, were equally distributed among patients differing in survival time. However, a remarkable acceleration was observed in the generation of abnormal cell lines in patients dying early of metastatic disease.