Characterization of an HPV-negative cell line (FR-CAR) derived from a cervical squamous intraepithelial lesion.

A new cell line, FR-car, has been established from a biopsy of a low-grade human cervical squamous intraepithelial lesion (SIL). We confirmed the epithelial origin of the cells by keratin staining using polykeratin, AE1/AE3 and CAM 5.2 antibodies. Sixty percent to 80% of the cultured cells stained positive for proliferative cell nuclear antigen (PCNA) and Ki-67. There was no overexpression of p53. Karyotyping revealed that the cell line was hypodiploid with clonal abnormalities on chromosome 6 and 16. Sections of a biopsy adjacent to the lesion from which the culture was initiated tested positive for human papillomavirus (HPV) 18 DNA by the polymerase chain reaction, but cultured cells tested at several passages were HPV-negative by either type-specific or consensus PCRs. This HPV-negative SIL line may be useful in studies into the cell biology of dysplastic epithelium.

Prognostic value of cytogenetic abnormalities in previously untreated patients with non-Hodgkin's lymphoma.

In this study cytogenetic findings have been correlated with prognosis in 78 previously untreated patients with non-Hodgkin's lymphoma (NHL) presenting between 1983 and 1988. The median follow-up was 7 years (range 2-9 years). There was no significant difference in the duration of survival of 33 patients with only abnormal karyotypes, 35 patients with a mixture of normal and abnormal karyotypes (AN) and 10 patients with only normal karyotypes (NN). This was true for the entire group (p = 0.6) as well as for the subsets of diffuse lymphomas (DL) and follicular lymphomas (FL) (p = 0.6 and 0.4, respectively). Monosomy 14 was the only abnormality in the entire group of patients to be associated with a statistically significant difference in survival duration (p = 0.046). Among the FL patients, trisomy 7 (p = 0.046) and trisomy 12 (p = 0.010) were associated with shorter survival. Presence of t(14;18) did not influence survival in the entire group (p = 0.16), nor in any of the histological subgroups. Among the FL patients with t(14;18), presence of additional cytogenetic abnormalities was not associated with a worse outcome. The lack of consistency of results between various studies is likely to be due to several factors and the prognostic significance of karyotypic abnormalities can only be clarified by large prospective studies employing uniform treatment policies.

Functional evidence for a colorectal cancer tumor suppressor gene at chromosome 8p22-23 by monochromosome transfer.

Chromosome 8p is considered, from loss of heterozygosity analysis, to be a strong candidate for the location of a tumor suppressor gene inactivated in colorectal cancer. We have found a 53% (27 of 51) rate of allelic loss at the LPL locus on 8p22, with the smallest region of overlap of deletions including the region D8S258 to D8S277. Using microcell-mediated monochromosome 8 transfer into three colorectal cancer cell lines, SW480, SW620 and HT29, we have demonstrated a reduction of tumorigenicity in SW620 hybrids. Partial deletions of chromosome 8 in some SW620/8 hybrids further delineate the critical region(s) to 8p22-23. Hybrids of the colorectal cancer cell lines SW480 and HT29 containing chromosome 8 did not show suppression of tumorigenesis, but the H29/8 hybrid showed total suppression of soft agar clonicity. This indicates an alternate pathway of mutational progression in these three lines, despite the fact that SW480 was derived from the same patient as SW620.

Association of abnormalities of chromosome 11 with t(14;18) in diffuse non-Hodgkin's lymphoma.

Cytogenetic studies of non-Hodgkin's lymphomas (NHL) have revealed a nonrandom translocation, t(14;18)(q32;q21), to be strongly correlated with follicular histology. In our recent study of 149 cases of NHL, 68 cases had a t(14;18). Forty-four of these were follicular and 24 diffuse. In the majority of cases (90%) there were additional chromosome abnormalities, which were analyzed to determine whether any were specifically associated with diffuse histology. Chromosome 11 abnormalities occurring together with the t(14;18) were found to be present in 17/68 cases; 14/17 (82%) were diffuse and 3/17 (18%) were follicular NHL. Thus, 14/24 (58%) of all diffuse lymphomas with t(14;18) had an abnormality of chromosome 11 compared to only 3/44 (7%) of follicular lymphomas, suggesting that the addition of an abnormality of chromosome 11 to a t(14;18) karyotype is associated with diffuse histology.

Characterization of homogeneously staining regions in a small cell lung cancer cell line, using in situ hybridization with an MYCN probe.

The cell line CIPL38 was derived from the pleural effusion of a patient with small cell lung cancer. The karyotype was hyperdiploid and complex with a variable number of marker chromosomes. Two of the markers had large homogeneously staining regions (hsr), which were shown to consist of amplified MYCN by in situ hybridization. One hsr bearing a marker chromosome could not be identified with G-banding, but the other was situated on a der(14). This was elucidated further with FISH analysis, which enabled the identification of sequences of chromosome i involved in a complex rearrangement with chromosome 14 and the hsr.

Karyotypic analysis of a heterogeneous human transitional cell carcinoma of the bladder.

The UCRU-BL-17 (BL-17) series of xenografts, tissue culture sublines, and cloned cell lines (Fig. 1) shows a range of heterogeneous growth characteristics both in vitro and in vivo (Table 1) and represents a model of human bladder cancer heterogeneity. Cytogenetic analysis was undertaken to determine if specific chromosome changes correlated with particular aspects of the heterogeneous phenotypes. The BL-17 sublines and cloned cell lines shared many common chromosome abnormalities. Indeed, the cloned cell lines showed nearly identical karyotypes despite their marked differences in growth characteristics. Karyotypic evolution with passage through the nude mice was apparent, however. This evolution occurred at the specific chromosome regions of 1p12, 3cen-3p21, and 6cen-6q21. Whether the heterogeneity of karyotype between the BL-17 cell lines resulted from the existence of multiple clones in the original patient tumor or from karyotypic instability through passage in nude mice is uncertain, but in either case the specificity of karyotypic evolution observed suggests that 1p12, 3cen-3p21, and 6cen-6q21 are hotspots for rearrangement in the BL-17 tumor. No specific correlations between chromosome abnormalities and biologic characteristics could be made, but several unique karyotypic features arose in the progression to two of the sublines, BL-17/23 alpha and BL-17/0/X2A, coinciding with a loss of anchorage-independent growth by BL-17/23 alpha and a change in growth in vivo from a solid tumor to a fluid-filled tumor by BL-17/0/X2A.

Chromosome abnormalities in non-small cell lung cancer pleural effusions: cytogenetic indicators of disease subgroups.

A cytogenetic study of pleural effusions (PE) containing metastatic or invasive tumor cells from 11 patients with non-small cell lung cancer (NSCLC) (3 squamous cell carcinomas [SQC] and 8 adenocarcinomas [ADC] including 1 giant cell variant) was performed to identify non-random chromosome abnormalities. Numerical abnormalities seen in > or = 30% of cases included gain of chromosomes 7 and 20, and loss of chromosomes 4, 9, 10, 13, 15, 16, 18, 19, 21, and 22. The most frequent structural abnormality involved rearrangement in 1p with breakpoints clustering at 1p10-p13. Other recurrent breakpoint regions, seen in > or = 30% of cases, occurred in chromosome region 3p10-p21, 3q11-q25, 6p11-p25, 6q13-q23, 7q11-q36, 9q32-q34, 11p11-p13, 11q13-q24, 13p/14p and/or 15p, 17p and 19p, with, in particular, apparent loss of 6q21-q27, 3p21-p26, 7q21-q22, 9p22-p24 (shortest regions of common overlap) and 17p. There was also recurrent gain of 1q23-q44, 8q13-q24, and 11q13-q23. These abnormalities were not restricted to a particular histological subtype, with the exception of +8 and a breakpoint in 9q32-q34, which were seen only in ADC. The 9q32-q34 breakpoint observed in 4 ADC PE (including 1 giant cell variant) represents a new observation in NSCLC. These findings, when compared to those reported for primary NSCLC indicate cytogenetic differences between the two which may be associated with pleural invasion of NSCLC.

Y chromosome loss and rearrangement in non-small-cell lung cancer.

While loss of the Y chromosome from the karyotype of tumor cells has frequently been found in a number of human malignancies of different types, structural alterations are a much less common finding. Prompted by the high frequency of cytogenetic Y chromosome loss found in primary non-small-cell lung cancer (NSCLC), and the fact that NSCLC karyotypes usually contain marker chromosomes of unidentified origin, we have determined the Y chromosome status of 12 NSCLC samples (7 cell lines and 5 primary tumors) at a molecular level. Of the 9 cases which did not have a cytogenetically detectable Y chromosome, 4 were negative for all the Y sequences tested. The other 5, in contrast, retained some Y chromosome sequences. In 1 case (H520), only Yq heterochromatic sequences were detected, whereas in the remaining 4 (L162, L93, L125 and L71) both Yq heterochromatic sequences and Y euchromatic sequences were retained. The region of common overlap for loss of Y euchromatin was Yp distal to the Y centromere. We hypothesize that deletion of Yp sequences may play a role in tumor progression in NSCLC due to loss of a tumor-suppressor gene.

Characterization of a new human bladder cancer cell line, UCRU-BL-28.

A new human bladder cancer cell line, UCRU-BL-28 has been established and characterized from a relapsed, cisplatin resistant, grade II, stage T4 tumor. This line is tumorigenic in nude mice and reflects the pathology of the original tumor. The morphology, the expression of tumor-associated antigens and EGF receptors, and the ability to grow both in an anchorage independent manner and in the absence of serum is explored. The BL-28 line has 71-74XXY chromosomes, with del 5q, der(9) and i(19q). Further studies on the molecular basis of bladder cancer, chemosensitivity to cisplatin, growth factor production and tissue invasion are under way.

Molecular deletion of 9p sequences in non-small cell lung cancer and malignant mesothelioma.

Previously we have reported non-random cytogenetic abnormalities involving the short arm of chromosome 9 (9P) in the majority of primary non-small cell lung cancer (NSCLC) patient samples, which indicated loss of DNA sequences. In another lung tumor, pleural malignant mesothelioma (MM), cytogenetic changes also include apparent deletions of 9p. To define the location and extent of deletions of 9p in NSCLC and MM, Southern blot analyses on six NSCLC and five MM cell lines using molecular probes to 9p loci (IFNA, IFNB1, D9S3, and D9S19) were performed, and DNA dosage was determined by densitometry. Our data demonstrated reduced dosage of 9p sequences in three of six NSCLC and four of five MM lines. A homozygous deletion of D9S3 was found in one NSCLC and one MM cell line. The region of common loss overlapped the D9S3 locus and was flanked by the IFNB1 and D9S19 loci. IFNB as previously been localized to 9p22, and the D9S3 and D9S19 loci have been mapped in this study by in situ hybridization to 9p21 and 9p13, respectively. We hypothesize the existence of one or more tumor suppressor genes on 9p with a role in the development or progression of NSCLC and MM.

Chromosomal localization of amplified N-myc in neuroblastoma cells using a biotinylated probe.

G-banded chromosome analysis of neuroblastoma cells from two children revealed homogeneously staining regions (hsr) in one patient and double minutes (dmin) in the other. Subsequently, both abnormalities were confirmed as areas of N-myc amplification using chromosomal in situ hybridization with a biotinylated N-myc probe. In addition, the first patient's karyotype contained a possible derivative chromosome 17, which was also demonstrated to contain amplified N-myc, indicating the presence of an hsr unidentified by G-banding. Intercellular heterogeneity in the degree of amplification was also identified in the nuclei of interphase cells. This technique provides a quick method for detecting gene amplification, the identification of which may have useful clinical implications.

A cell line (LIM 2463) derived from a tubulovillous adenoma of the rectum.

We describe a new epithelial cell line (LIM 2463) derived from tubulovillous adenoma of the rectum. The cells grow as organoids and secrete large amounts of mucus. The cells are polarized, with a microvillar brush border, and express dipeptidyl peptidase IV in a polarized manner. No staining was seen with 3 other antibodies directed against other brush-border hydrolases or disaccharidases. Focal polarized staining was obtained with 2 antibodies directed against other brush-border-associated peptides. The cells are aneuploid with a distinctive karyotype (48, XX, +9, +13). All attempts to clone the cells in semi-solid agar or to grow them as xenografts in nude athymic mice have failed.

Cytogenetic analysis of 147 cases of non-Hodgkin's lymphoma: non-random chromosomal abnormalities and histological correlations.

A prospective cytogenetic study of patients with non-Hodgkin's lymphoma (NHL) presenting to one institution was commenced in 1983 as part of a larger study including histology, immunophenotyping, cytokinetics and survival. 175 patients were studied over 5 years and G-banded karyotypes were successfully obtained in 147. Chromosome abnormalities were detected in 135 cases (92%) with the commonest abnormality being t(14;18)(q32;q21) in 69 cases. Other non-random translocations were much less frequent, i.e. t(11;14) in seven cases and t(8;14) in four cases. Other specific structural changes included partial deletions of 6q (breakpoints ranging within q13-q23), 3q (breakpoints ranging within q21-q27), 1q and 10q22. Chromosome regions highlighted as being frequently involved in structural abnormalities were 11q13-q25, 1p22-p36, 3q21-q27 and 6q13-q23. Several specific recurring breakpoints were identified and these included 14q32, 18q21, 1p36 and 6q21. Frequently occurring numerical abnormalities were gains of chromosomes 3, 7, X and 12. Correlation with histological type showed, as expected, that t(14;18) was present in 89% of follicular lymphoma but also occurred in 30% of diffuse lymphoma. Abnormalities of 11q were correlated with the diffuse histologies as a group, whereas both numerical and structural abnormalities of chromosome 3 correlated with the diffuse large cell lymphoma (DLCL) subtype, and t(11;14) with diffuse small cleaved cell lymphoma (DSCCL). Although not statistically significant, abnormalities of 6q occurred twice as frequently in DLCL than in any other variety. However, several other commonly occurring abnormalities, such as extra copies of chromosomes 7, X, 12 and most of the structural abnormalities of 1p, did not correlate with any histological type. Therefore this large cytogenetic study has confirmed some previously reported correlations between specific chromosome abnormalities and histological subtypes of non-Hodgkin's lymphoma and has also identified some new correlations which may prove useful in the investigation of the biological basis of the disease.

Cytogenetics of non-small cell lung cancer: analysis of consistent non-random abnormalities.

Cytogenetic analysis of ten primary non-small cell lung carcinomas (NSCLC), including five adenocarcinomas (ADC), three squamous cell (SQC), and two large cell (LCC) carcinomas has been carried out in an attempt to determine karyotype changes involved in the early stage of disease. The tumors were all aneuploid and exhibited complex karyotypes with multiple structural and numerical abnormalities. Clonal structural rearrangements were identified and in particular loss of material from the short arm of chromosome 9 had a 90% incidence. This loss was due to non-reciprocal translocation, deletion, or chromosome loss. Breakpoints were in the region 9q13 to p22. Other chromosome regions that were non-randomly involved are as follows: I cen to p13, 3p, 5q11 to q13, 6p, 6q15 to q27, 7p, 8p, 11q12 to q23, 13p, 14p, 15p, 17p, and 19p. While a primary cytogenetic change in NSCLC has not been identified conclusively, our findings implicate loss of material from 9p as a potentially important event.

Establishment and characterization of a childhood pre-B acute lymphoblastic leukemia cell line, PER-278, with chromosome translocations t(1;19) and t(1;9).

Cell line PER-278 was established from a bone marrow sample of a 10-year-old boy diagnosed with pre-B acute lymphoblastic leukemia (ALL). PER-278 cells show the pre-B phenotype, express cytoplasmic Ig, and exhibit two translocations: t(1;19)(q23;p13) and t(1;9)(q23;p13). Assessment of the immunoglobulin rearrangements confirmed the clonal origin of cell line PER-278, and comparison with the patients's leukemic cells showed an identical pattern: loci involved at the breakpoint on chromosome 1 code for the oncogene SKI and for the Fc receptor II and on chromosome 19 for the insulin receptor. The t(1;19) may contribute to the malignant transformation in leukemic cells of pre-B phenotype.

A new gastric carcinoma cell line (LIM1839) derived from a young Caucasian male.

We describe a new and unique gastric carcinoma cell line (LIM1839) derived from a young Caucasian male with rapidly progressing disease. The cell line grows with a pleomorphic morphology and has been in continuous culture for more than 3 years. The cells cannot be cloned in semi-solid agar or grown in nude mice despite numerous attempts. The karyotype of the cultured cells is highly abnormal with a large number of structural and numerical changes. Some chromosomes are dicentric and this feature has persisted in this culture. The cells express one of the small-intestinal dipeptidases, aminopeptidase N, but do not express dipeptidyl peptidase IV or the disaccharidases, sucrase isomaltase or maltase glucoamylase. The cells express high levels of EGF receptors and of messenger RNA for insulin-like growth factor II.

Characterization of cell lines derived from a multiply aneuploid human bladder transitional-cell carcinoma, UCRU-BL-13.

A series of cultured cell lines (designated UCRU-BL-13) has been established from different serial passages of a multiply aneuploid human bladder transitional-cell carcinoma xenografted in nude mice. Serial passage of the xenografts in vivo and of the cell lines in vitro was accompanied by shifts in the tumor ploidy, with dominance of different major peaks. Despite this, the expression of tumor markers remained constant, and consistent chromosomal markers were observed both in the 8th xenograft passage and in a subline in tissue culture established over a year apart. Chromosomal numbers reflected the predominant aneuploid peaks observed; consistent numerical and structural changes included a marker derived from chromosome 1, 8p-, -10, 11q+, and 17q+. The cell line derived from the initial xenograft comprised a mixture of transitional, adenocarcinoma and squamous carcinoma cells in early passage, but adenocarcinoma cells were absent from later passages. The lines expressed the B-blood-group antigen, histocompatibility antigens, receptors for transferrin and EGF, and reacted with a series of monoclonal antibodies (MAbs) directed to malignant human epithelial cell lines. These lines provide a model for studying the evolution of tumor heterogeneity and drug resistance in bladder carcinoma exhibiting multiple aneuploidy.

Establishment and characterization of a childhood T-cell acute lymphoblastic leukemia cell line, PER-255, with chromosome abnormalities involving 7q32-34 in association with T-cell receptor-beta gene rearrangement.

A human leukemia cell line, PER-255, was established from the bone marrow of a 5-year-old boy with features typical of lymphomatous T-acute lymphoblastic leukemia (T-ALL). The leukemic origin of cell line PER-255 is indicated by its cytochemical and immunologic similarity to the patient's fresh leukemic cells, which correspond to immature cortical thymocytes. Southern blot analysis showed that the IgJH genes were in germline configuration, whereas both alleles of the T-cell receptor-beta (TCR-beta) gene were rearranged in PER-255 cells, with identical rearrangements present in the patient's leukemic cells. Cytogenetic analysis of the cell line revealed a single abnormal clone with the karyotype 46,XY,t(7;10)(q32-34;q24),t(9;12) (p22;p12-13). Reciprocal translocations involving chromosome bands 7q32-36, containing the gene for the TCR-beta chain, have been reported for a number of tumors of T-cell origin. Translocations involving the 7q32-36 region appear to be nonrandomly associated with childhood T-ALL, whereas abnormalities of 9p and 12p have been reported to be nonrandomly involved in ALL but not specifically associated with the T-cell phenotype.

Chromosome studies in inherited nonpolyposis colon cancer syndrome.

This study was designed to determine if any constitutional chromosomal markers were linked with the expression of colorectal neoplasms in the inherited nonpolyposis colon cancer syndrome, using a number of cytogenetic techniques. High resolution G-banding in 12 affected and 17 unaffected family members did not reveal a structural chromosome abnormality. Increased C-band heteromorphism was not seen in either affected or unaffected individuals, and no heritable fragile sites were detected. Mean baseline and mitomycin C-induced sister chromatid exchanges were not elevated in affected patients compared with controls. Mapping of sister chromatid exchanges did not reveal any hot spots of exchange. A tumor cell line with the karyotype 46,XY,der(13),t(13;?)(p11;?) was established from one patient, but no constitutional abnormality of chromosome #13 was found. In addition, 11 patients with familial polyposis coli were studied with high resolution G-banding and no heteromorphism of chromosome #2 in the region 2q21.3 was detected.