Analytical evaluation of the new Prostatus PSA Free/Total assay for prostate-specific antigen as part of a screening study for prostate cancer

A second Philadelphia (Ph) chromosome is one of the most common nonrandom secondary chromosome changes in leukemias with 9;22 translocations. It has been suggested, and observed in two studies of masked t(9;22), that the second Ph chromosome is an exact duplication of the entire derivative chromosome 22. In a cytogenetic study of bone marrow cells from an acute myelogenous leukemia patient, a cell line carrying two different Ph chromosomes evidenced by a chromosome 22 centromeric heteromorphism was found. From this observation arose the question whether the second der(22) was a true Ph chromosome or whether it was a deleted chromosome derived from the normal chromosome 22 that did not contain the bcr-abl rearrangement. A fluorescent in situ hybridization (FISH) study with the t(9;22) probe revealed two bcr-abl positive signals on 60 of 100 interphase nuclei. The second Ph could have resulted from a mitotic crossing over; or, analogously to late-appearing Philadelphia chromosomes, it may be derived from a new chromatid translocation between the chromosomes 9 and 22 not involved in the initial t(9;22).     

Preliminary mapping of the deleted region of chromosome 9 in bladder cancer

Inactivation of a suppressor gene by deletion of chromosome 9 is a candidate initiating event in bladder carcinogenesis. We have used 13 polymorphic markers spanning the length of chromosome 9 in order to map the region of deletion in human bladder carcinomas. In the majority of tumors loss of heterozygosity was found at all informative sites along the chromosome, indicating deletion of the entire chromosome. Nine tumors had selective deletions of chromosome 9. Mapping of the deleted region in these tumors suggests that the target gene is located between D9S22 at 9q22 and D9S18 at 9p12-13.     

Is chromosome 9 loss a marker of disease recurrence in transitional cell carcinoma of the urinary bladder?

Investigation of transitional cell carcinoma of the urinary bladder (TCC) patients classified by recurrence and/or progression has demonstrated that loss of chromosome 9, as detected by FISH analysis of the pericentromeric classical satellite marker at 9q12, occurs early. A total of 105 TCCs from 53 patients were analysed in situ by two independent observers for loss of chromosome 9 using quantitative fluorescence in situ hybridization (FISH). All 53 primary tumours were evaluated for chromosomes 9, 7 and 17. Normal ranges for chromosomal copy number were defined for normal skin epidermis and bladder epithelium. Values for chromosome 9 copy number outwith the range 1.51-2.10 (mean +/- 3 x s.d. of normal values) were significantly abnormal. Twenty-five TCCs were detected with consistent monosomic scores. Of 89 TCCs, in which multiple tumour areas were analysed, 85 tumours (96%) demonstrated the same chromosome 9 copy number in all areas (2-6) analysed; only three tumours demonstrated heterogeneity for this locus. A total of 36% (12 out of 33) of patients with subsequent disease recurrence demonstrated loss of chromosome 9 in their primary and all subsequent TCCs analysed. Only a single patient (n = 20) with non-recurrent TCC showed loss of chromosome 9 (P = 0.0085). Of 53 primary tumours, eight showed significant elevation of chromosome 17. Of these patients, six demonstrated elevation in chromosome 7 copy number. No abnormalities were observed in non-recurrent patients. This study describes rapid quantitation of chromosomal copy number by FISH using a pericentromeric probe for chromosome 9 in TCC of the urinary bladder. Routinely fixed and processed material was evaluated without disaggregation. Strict quality control of FISH demonstrated that this technique was reproducible in a clinical environment and could be used to detect genetic changes relevant to patient outcome. It is proposed that loss of chromosome 9 from primary TCC of the urinary bladder identified patients at high risk of recurrence and possible progression.     

A case with dicentric translocation between chromosome 9 and 18: confirmation by fluorescent in situ hybridization on metaphase spread

A female child with dicentric translocation between chromosome 9 and chromosome 18 presented non-specific minor anomalies with laryngomalacia. Chromosomal analyses were performed by the G-banding method and a fluorescence in situ hybridization (FISH) technique with a specific probe for the centromeric region of chromosome 18 and the painting probe for the chromosomes 9 and 18. Her full karyotype was confirmed as 45,XX,tdic(9;18)(p24;p11). This is the first case of dicentric translocation between chromosomes 9 and 18. The FISH technique is an important tool in chromosome diagnostics.     

Homozygosity for pericentric inversions of chromosome 9. Prenatal diagnosis of two cases

The finding of homozygosity for a pericentric inversion of chromosome 9 [inv(9)] is rare, and previously has not been reported at prenatal diagnosis. We describe two unrelated cases of homozygosity for inv(9) identified in amniocytes. In each case, both parents were heterozygotes for the inv(9); 46,XX,inv(9)(p11q13) and 46,XY,inv(9) (p11q13). Case 1 resulted in a normal term infant who at age 5 years was phenotypically and developmentally normal. Case 2 was referred for severe intrauterine growth retardation (IUGR) and oligohydramnios, and subsequently expired in utero. Even though inv(9) is a normal chromosome variant with a frequency of 1 to 3% in the general population, the finding of homozygosity for inv(9) and IUGR in this fetus suggested the possibility of uniparental disomy (UPD). Molecular studies confirmed the presence of both parental inv(9) chromosomes, excluding the possibility of chromosome 9 UPD as the cause of IUGR in this fetus. Presumably, inv(9) homozygosity results from the high frequency of inv(9) heterozygosity, and is a normal variant. However, until the effects of UPD for chromosome 9 are established, parental karyo types and, where appropriate, molecular studies should be performed to exclude UPD. In addition, more reports of inv(9) homozygosity detected prenatally are needed to assess its frequency and outcome.     

A case of transitional cell carcinoma of the bladder with a del(9)(q11q21.2)

Monosomy for chromosome 9, as well as loss of heterozygosity for markers on this chromosome, has been detected in a high percentage of transitional cell carcinomas (TCC) of the bladder. We report a case of a TCC of the bladder with an interstitial del(9)(q11q21.2) that could be indicative of the presence of a putative tumor-suppressor gene related to bladder tumor progression. To elucidate the role of chromosome 9 in bladder tumors, it would be interesting to study a possible loss of heterozygosity in this chromosome region.     

Suppression of tumorigenicity and induction of senescence on human endometrial carcinoma cell lines by transfer of normal human chromosomes

The author examined the ability of human chromosomes derived from normal fibroblast cells to suppress the tumorigenicity of HHUA and Ishikawa cells, human endometrial carcinoma cell lines. Using DNA transfection, the human chromosome tagged with a selectable marker (the pSV2neo gene, which encodes resistance to the antibiotic, G418) was transferred to mouse A9 cells by cell hybridization and microcell fusion techniques. Thus, a library of mouse A9 clones containing individually a different human chromosome tagged with the pSV2neo plasmid DNA was constructed. Transfer by microcell fusion of either chromosome 1, 6, 9, 11 or 19 into the HHUA and Ishikawa cell lines was performed, and the abilities of the microcell hybrids to form tumors in nude mice were examined. The introduction of chromosome 19 had no effect on the tumorigenicity, whereas microcell hybrid clones with an introduced chromosome 1, 6 and 9 completely suppressed the tumorigenicity of the both lines. A decrease in tumor-take incidence in some but not all clones of HHUA cells was observed following the introduction of a chromosome 11. The nontumorigenic microcell hybrids with an introduced chromosome 1 differed from the nontumorigenic microcell hybrids with an introduced chromosome 6, 9, or 11. A large percentage of hybrids with chromosome 1 sensed and/or showed alterations in cellular morphology and transformed growth properties in vitro on the both cell lines. These results indicate that more than one chromosome carries a tumor suppressor gene(s) for human endometrial carcinoma cell lines, and indicate that normal human chromosome 1 carries gene(s) which suppresses the immortalization. This supports the hypothesis that multiple tumor suppressor gene(s) control the various tumorigenic phenotypes at the different step during process of neoplastic development.     

Duplication of 9P and hyperplasia of the choroid plexus: a pathologic, radiologic, and molecular cytogenetics study

Duplication of the short arm of chromosome 9 is a rare constitutional abnormality, and the presence of pathologically confirmed hyperplasia of the choroid plexus in one of two cases, and a choroid plexus papilloma in another, raises the possibility of a relationship between the 9p abnormality and abnormal growth of the choroid plexus. Molecular cytogenetic analysis using fluorescence in situ hybridization was used for detection of chromosome 9-derived material in various formalin-fixed choroid plexus abnormalities. Extra copies of chromosome 9-derived material was found in the hyperplastic choroid plexus and in a choroid plexus carcinoma. These findings suggest that there may be an association between duplication of chromosome 9 material and abnormal development of the choroid plexus.     

Genetic and physical mapping of telomeres and macrosatellites of rice

Telomeres and telomere-associated satellites of rice were genetically and physically analyzed by pulsed-field gel electrophoresis (PFGE) using Arabidopsis telomeric DNA and rice satellite sequences as probes. We demonstrate that Arabidopsis telomeric sequences hybridize to rice telomeres under the conditions of high stringency. Using the Arabidopsis probe, multiple, discrete telomeric fragments could be identified on pulsed-field gel blots of rice DNAs digested with rare-cutting restriction enzymes. Most of the telomeric bands larger than 300 kb are physically linked with satellite bands as revealed by PFGE. Some of the telomeric and satellite bands segregate in a Mendelian fashion and are highly reproducible. Three such telomeric bands have been mapped to the distal ends of RFLP linkage groups: Telsm-1 on chromosome 8, Telsa-1 on chromosome 9 and Telsm-3 on chromosome 11. One segregating satellite band was mapped to an internal region of chromosome 10. Telomeric fragments were shown not only to be genetically linked to but also physically linked (based on PFGE) to the terminal RFLP markers. The physical distance from telomeric sequences to a distal RFLP marker, r45s gene, on chromosome 9, is 200 kb while the distance from telomeric sequences to RG98, a terminal RFLP marker on chromosome 11, is 260 kb. Physical maps of the telomere regions of chromosome 9 and chromosome 11 are presented.     

Genomic organization and mutation analysis of Hel-N1 in lung cancers with chromosome 9p21 deletions

Allelic loss of chromosome 9p21 is common in small cell lung cancer (SCLC), but inactivation of the tumor suppressor gene CDKN2a is rare, implying the existence of another target gene at 9p21. A recent deletion mapping study of chromosome 9p has also identified a site of deletion in non-small cell lung cancer (NSCLC) centered around D9S126. The Hel-N1 (human elav-like neuronal protein 1) gene encodes a neural-specific RNA binding protein that is expressed in SCLC. We have mapped this potentially important gene in lung tumorigenesis to within 100 kb of the D9S126 marker at chromosome band 9p21 by using homozygously deleted tumor cell lines and fluorescence in situ hybridization to normal metaphase spreads. Hel-N1 is, therefore, a candidate target suppressor gene in both SCLC and NSCLC. We have determined the genomic organization and intron/exon boundaries of Hel-N1 and have screened the entire coding region for mutations by sequencing 14 primary SCLCs and cell lines and 21 primary NSCLCs preselected for localized 9p21 deletion or monosomy of chromosome 9. A homozygous deletion including Hel-N1 and CDKN2a was found in a SCLC cell line, and a single-base polymorphism in exon 2 of Hel-N1 was observed in eight tumors. No somatic mutations of Hel-N1 were found in this panel of lung tumors. Hel-N1 does not appear to be a primary inactivation target of 9p21 deletion in lung cancer.     

Evidence of chromosome 9 origin of the euchromatic variant band within 9qh

Fluorescence in-situ hybridization studies using a whole chromosome 9 painting probe were performed on three individuals from two different families, who carry a chromosome 9 variant with an extra band within the elongated 9qh region. The results confirm the euchromatic nature of the extra band, and provide evidence that it is of chromosome 9 origin. This variant band, which may not be very rare, thus possibly results from a duplication of a segment of 9qh plus band p12 or part of band q21.     

Detailed deletion mapping of the long arm of chromosome 6 in adult T-cell leukemia

Previously, we have found that the loss of heterozygosity (LOH) was frequently observed on chromosome 6q in acute/lymphoma-type adult T-cell leukemia (ATL), suggesting a putative tumor-suppressor gene for ATL may be present on chromosome 6q. To further define a region containing this gene, we performed fine-scale deletional mapping of chromosome 6q in 22 acute/lymphomatous ATL samples using 24 highly informative microsatellite markers. LOH was found in 9 samples (40. 9%) at 1 or more of the loci examined. Of the 9 samples, 8 shared the same smallest commonly deleted region flanked by D6S1652 and D6S1644 (6q15-21). The genetic distance between these two loci is approximately 4 cM. These results suggest that a putative tumor-suppressor gene on chromosome 6q15-21 probably plays a very important role in the evolution of acute/lymphomatous ATL. Our map provides key information toward cloning the gene.     

A fatal case of intrathoracic cuterebriasis in a cat

Isolates of Plasmodium falciparum commonly undergo a large, subtelomeric deletion of the right end of chromosome 9 during in-vitro cultivation. This deletion is usually accompanied by loss of ability to cytoadhere to melanoma cells, loss of a var-gene product from the red-cell surface and a reduction in gametocyte production. However, cytoadherence is stable in the isolate ItG2, remaining after many generations in culture. Deletions in all the non-cytoadherent clones examined have breakpoints within or delete a novel open-reading frame, called the breakpoint open-reading frame (BPORF), that is a unique sequence in the genome. In ItG2, surprisingly, BPORF has been removed by a 15-kb deletion, internal in chromosome 9. These results indicate mechanisms to explain why the deletion of chromosome 9 occurs so frequently and why cytoadherence is stable in ItG2.     

A case of double primary adenocarcinoma of the lung with multiple atypical adenomatous hyperplasia

A case of double primary adenocarcinoma of the lung with multiple atypical adenomatous hyperplasia (AAH) in a 77-year-old woman is reported. Histopathologically, in the resected left upper lobe of the lung, both cancers were diagnosed as well-differentiated papillary adenocarcinoma, and 161 lesions of AAH were also found. Both the cancer lesions and six AAH (greater than 3 mm in diameter) were examined with regard to immunoreactivity of carcinoembryonic antigen (CEA) and p53 gene product, microsatellite instability (MI) and loss of heterozygosity (LOH) on chromosome 9q and 17q by polymerase chain reaction (PCR). Although both cancers expressed CEA, they did not show clonal immunoreactivity for the p53 gene product. Atypical adenomatous hyperplasia expressed CEA weakly and showed no immunoreactivity for p53 gene protein. Both carcinomas showed LOH on chromosome 17q, and one of them showed LOH on chromosome 9q. In six AAH, LOH on chromosome 17q was detected in two tumors, and one of them also showed LOH on chromosome 9q. One AAH, which was negative for LOH on chromosome 17q and 9q, showed MI at D17S791. These results indicated that AAH is a clonal neoplastic lesion with genetic abnormalities and should be called intraepithelial pneumocyte neoplasia, and that each of the numerous papillary lesions in this case was considered to be an independent lesion.     

Endogenous retroviral sequences analogous to that of the new retrovirus MSRV associated with multiple sclerosis (part 1)

Human lymphocyte cultures were treated with iododeoxyuridine. This 5-halogenated thymidine analogue induces distinct undercondensations of the heterochromatin of human chromosome 9. The condensation of the other heterochromatic regions on chromosomes 1, 15, 16 and Y is also inhibited, but to a lesser extent. Optimum cell culture conditions required for the induction of undercondensations were determined. Up to 90% of mitotic cells reveal chromosome 9 heterochromatin decondensations when the substance is present in quantities of 1 x 10(-4) M during the last 7 h before cell fixation. In addition, examples of the usefulness of 5-IUDR in the analysis of chromosome aberrations involving chromosome 9 are presented. The interaction between 5-IUDR and chromosomal DNA, the modification of chromosomal proteins and factors inducing chromosomal decondensations are discussed.     

A rapid and selective endothelin-converting enzyme assay: characterization of a phosphoramidon-sensitive enzyme from guinea pig lung membrane

A linkage map of pig chromosome 1 (SSC1) has been constructed using 15 polymorphic markers. Eleven markers constitute PCR-based microsatellites (three associated with coding sequences), six markers have also been mapped by in situ hybridization, and homologues to four of the markers have been mapped in human. The physical assignments show that almost the entire SSC1 is covered by the linkage map, which measures 164 cM (sex-averaged). In a large region on the q arm, representing about 40% of the chromosome, there is a significant excess of male recombination. In contrast, there is a significantly higher recombination rate in females in both terminal regions. In the penultimate marker intervals on the q arm as well as the p arm, females show a fivefold excess of recombination compared to males. The rate of genetic recombination in relation to the physical DNA content is 1 cM per 2-4 Mb over at least 80% of the chromosome. In the terminal part of the q arm, however, there is a tremendous increase in the recombination rate, with 1 cM equaling about 0.4 Mb. Two homologous chromosome segments in human could be detected, ESR-CGA on human chromosome 6 (HSA6) and IFNA-RLN-GRP78 on human chromosome 9 (HSA9). Since the porcine blood group locus EAA is located close to (or possibly within) the latter conserved segment and the suggested human counterpart, the ABO system, is similarly close to the segment on HSA9, our data provide indirect evidence that these blood group systems are homologous.     

Frequent loss of heterozygosity on chromosome 9 in adenocarcinoma and squamous cell carcinoma of the esophagus

Loss of heterozygosity (LOH) affecting chromosome 9p has been shown to occur frequently in head and neck cancer, glioma, mesothelioma, melanoma, lung cancer, and numerous other tumor types. Chromosome 9p is therefore presumed to contain a tumor suppressor gene or genes. Since esophageal cancer shares characteristics with some of the above tumor types, we performed a detailed examination of 60 patients with squamous cell carcinoma or adenocarcinoma of the esophagus for LOH at loci D9S162, IFNA, D9S171, D9S126, D9S104, D9S165, and D9S163. Multiplex polymerase chain reactions were performed with the inclusion of one radiolabeled nucleotide, and products were electrophoresed on denaturing polyacrylamide gels. Thirty-six of the 60 patients (60%) exhibited LOH at one or more loci on chromosome 9p. Eight of 17 patients (47%) with adenocarcinoma manifested LOH, while 28 of 43 (65%) with squamous cell carcinoma showed LOH. LOH was most frequent at loci D9S171 (19 of 23, or 83%) and D9S165 (24 of 32, or 75%). These data support the hypothesis that a tumor suppressor gene or genes located on this portion of chromosome 9p exert(s) an effect on esophageal cancer development.     

Variants of chromosome 9 in phenotypically normal individuals

The human chromosome 9 displays the highest degree of structural variability. Four different types of variants are described including pericentric inversion, extra G-positive band in the q arm, additional G-positive band in the p arm and duplication of band 9q21-q22. It is important to demonstrate inheritance from a phenotypically normal individual in order to differentiate between a variant chromosome and an abnormal chromosome.     

Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon gamma receptor: evidence for multiple accessory factors

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-gamma, and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hu-IFN-gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. Here we report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity.     

Allelic loss on chromosome 9q is associated with lymph node metastasis of primary breast cancer

Frequent allelic losses on chromosome 9 are seen in a wide variety of human tumors; moreover, two genes (P16 and PTC) whose mutant alleles confer predispositions to some inherited cancer syndromes have been identified on this chromosome. Using 15 highly polymorphic microsatellite markers distributed on both arms of chromosome 9, we tested 96 primary breast carcinomas for allelic loss in order to define the locations of genes that might be involved in this type of tumor. Allelic loss was observed in 37 of the tumors (39%) and detailed deletion mapping identified target regions at 9p21, 9q22.3 and 9q33. Losses at 9q22.3 and 9q33 were correlated with the presence of lymph node metastasis, and allelic loss at 9q22.3 was observed more frequently in scirrhous tumors than in less aggressive histologic types. Therefore, inactivation of tumor suppressor genes in 9q22.3 and 9q33 regions might play a role in progression of breast cancers, especially in metastasis to lymph nodes and in development of scirrhous tumors.