Fluorescent banding patterns of rat chromosomes in normal cells and primary hepatocellular carcinomas.

Quinacrine staining permits identification of rat chromosomes in metaphase that were formerly classified only in groups within the karyotype. This technique defined two types of abnormal chromosomes in cells of rat hepatomas.

Evidence for the clonal origin of spontaneous metastases.

A cultured cell line of the K-1735 melanoma was x-irradiated to induce chromosome breakage and rearrangements and then was implanted into the footpads of syngenic C3H mice. Spontaneous lung metastases were isolated from different animals, established in culture as individual lines, and then karyotyped. Within certain metastases, the same chromosomal abnormality (or abnormalities) (recombinant chromosomes) was found in all the cells examined. Most metastases differed from one another in that they exhibited characteristic combinations of chromosomal markers. These findings indicated that the metastases were clonal and that they probably originated from different progenitor cells.

Amplification of the c-myb oncogene in a case of human acute myelogenous leukemia.

Amplification is one of the mechanisms by which cellular oncogenes may be altered in their function, possibly leading to neoplastic transformation. The oncogenes c-myc, c- abl , and c-Ki-ras are amplified in several different human neoplasias. The oncogene c-myb, which is specifically expressed and regulated in hematopoietic cells, was found to be amplified in cell lines ML-1, ML-2, and ML-3, which were separately cultured from cells of a patient with acute myelogenous leukemia (AML). A five- to tenfold amplification was correlated with high levels of expression of normal size c-myb messenger RNA and with chromosomal abnormalities in the region 6q22 -24, where the c-myb locus is normally located. Amplification and cytogenetic abnormalities were detected in DNA's from primary and secondary cultures of ML cells, suggesting that they may have contributed to leukemogenesis. The similar AML cell lines HL-60 and ML's contain different amplified oncogenes: c-myc and c-myb, respectively. Alternative activation of structurally and possibly functionally similar oncogenes may distinguish--at the pathogenetic level--phenotypically similar tumors.

Immortalization in culture: occurrence at a late stage in the progression of breast cancer.

The properties in culture of 3 breast cancer effusion metastases, obtained over approximately 2 years from the same patient, were examined. Despite repeated attempts with cryopreserved cells, only the last specimen reproducibly exhibited immortality in culture; the first 2 specimens grew initially but failed to develop into cell lines. Each specimen was unique in morphology and growth properties, although karyotypic markers indicated a common origin. Aberrations of chromosomes 1 and 11 marked these near-diploid cells, and further structural alterations of chromosome 11 accompanied the transition of biological properties observed in the third specimen.

Race and differences in breast cancer survival in a managed care population.

BACKGROUND: African-American women with breast cancer have poorer survival than European-American women. After adjustment for socioeconomic variables, survival differences diminish but do not disappear, possibly because of residual differences in health care access, biology, or behavior. This study compared breast cancer survival in African-American and European-American women with similar health care access. METHODS: We measured survival in women with breast cancer who are served by a large medical group and a metropolitan Detroit health maintenance organization where screening, diagnosis, treatment, and follow-up are based on standard practices and mammography is a covered benefit. We abstracted data on African-American and European-American women who had been diagnosed with breast cancer from January 1986 through April 1996 (n = 886) and followed these women for survival through April 1997 (137 deaths). RESULTS: African-American women were diagnosed at a later stage than were European-American women. Median follow-up was 50 months. Five-year survival was 77% for African-American and 84% for European-American women. The crude hazard ratio for African-American women relative to European-American women was 1.6 (95% confidence interval [CI] = 1.1-2.2). Adjusting only for stage, the hazard ratio was 1.3 (95% CI = 0.9-1.9). Adjusting only for sociodemographic factors (age, marital status, and income), the hazard ratio was 1.2 (95% CI = 0.8-1.9). After adjusting for age, marital status, income, and stage, the hazard ratio was 1.0 (95% CI = 0.7-1.5). CONCLUSION: Among women with similar medical care access since before their diagnoses, we found ethnic differences in stage of breast cancer at diagnosis. Adjustment for this difference and for income, age, and marital status resulted in a negligible effect of race on survival.

Xenograft model of progressive human proliferative breast disease.

BACKGROUND: Progression of proliferative breast disease has been associated with increased risk for development of invasive carcinoma. Cell lines have been developed to facilitate the study of this process. Human cell line MCF10A originated from spontaneous immortalization of breast epithelial cells obtained from a patient with fibrocystic disease, and cell lines MCF10AneoN and MCF10AneoT were created by stable transfection of these cells with the neomycin-resistance gene and either the HRAS gene or the mutated T-24 HRAS gene, respectively. PURPOSE: Our goal was to develop an experimental model of progressive human proliferative breast disease. METHODS: MCF10A, MCF10AneoN, and MCF10AneoT cells were injected subcutaneously into the dorsal flank of male nude/beige (C57/BALB/c nu/nu bg/bg) mice (12 mice for each cell type). These mice were examined periodically for formation and persistence or growth of palpable nodules. One mouse per group was killed 1 week after cell injection; thereafter, mice were observed as long as possible. Cells were recovered from palpable lesions by enzymatic dissociation of the excised lesions. Cells re-established in tissue culture from a week-14 tumor (MCF10AneoT.TG1) were injected into 12 male nude/beige mice. Southern blot hybridization analysis of the HRAS gene locus and cytogenetic analyses were performed. RESULTS: Transplanted MCF10A and MCF10AneoN cells formed transient, small palpable nodules that regressed and disappeared during the 4th and 5th weeks. In 10 of the 12 mice, T-24 HRAS gene-transfected MCF10A cells (MCF10AneoT) formed small, flat nodules that persisted for at least 1 year. Three of these xenografts became carcinomas. One (removed 7 weeks after transplantation) was an undifferentiated carcinoma composed of polygonal cells with large, vesicular nuclei and numerous mitoses. The second (removed after 14 weeks) was an invasive squamous cell carcinoma. The third (removed after 56 weeks) was a moderately differentiated adenocarcinoma. Initially, xenografts of MCF10AneoT.TG1 cells showed intraductal proliferative changes; after 23 weeks, the lesions showed histologic features resembling those seen in atypical hyperplasia of the human breast, and later lesions showed characteristics of carcinoma in situ. The MCF10 lineage of cells of three MCF10AneoT.TG1 xenografts was confirmed by DNA fingerprinting and karyotype analysis. CONCLUSIONS: MCF10AneoT and MCF10AneoT.TG1 comprise a transplantable xenograft model that produces a broad spectrum of human proliferative breast disease. IMPLICATIONS: The reproducible establishment of representative stages in early breast cancer progression from the MCF10 model offers a new opportunity to analyze critical events of carcinogenesis and progression in breast cancer.

Xenografts of primary human prostatic carcinoma.

BACKGROUND: Prostatic carcinoma is both the most common invasive cancer and the second most common cause of cancer deaths in men in the United States. Before 1991, attempts to propagate prostatic carcinoma from primary tumors for periods longer than 3 months were unsuccessful in vivo and in vitro with rare exceptions. In 1991, we reported establishment of slowly growing tumors for six of 10 human primary prostatic carcinomas approximately 2-6 months after transplantation. However, none of the tumors were larger than 5 mm or serially transplantable. PURPOSE: Our purpose in this study was to determine whether human primary prostatic carcinoma could be grown as serially transplantable xenografts. METHODS: Cells from primary prostatic carcinomas obtained from transurethral prostatic resections or total prostatectomies in 20 patients were injected subcutaneously into male nude mice on the day of surgery. Sustained-release testosterone pellets were placed subcutaneously in the mice 2-24 days before transplantation of tumors and at intervals of 10-12 weeks. Serial transplantations in subsequent generations of mice were carried out by similar methods. Chromosome analysis was performed on six tumors. RESULTS: Six of 20 primary prostatic carcinomas have grown sufficiently to permit serial transplantation into second mice; four have been documented histopathologically in the second mouse and serially transplanted into three or more successive mice. When a single primary tumor was injected into several mice by the same procedure, tumors failed to grow in some recipients but became serially transplantable in others. Growth of these tumors is slow and irregular, with frequent regressions. Short-term cultures of 10 tumors, eight of which were injected into mice in parallel, were initiated on the day of surgery; CWR31, which was successfully transplanted serially, exhibited only aberrant metaphases and showed clonal, chromosomal changes in culture. Including CWR31, three of the six tumors for which chromosomal analysis was successful contained clonal aberrations. Preliminary studies of SCID (severe combined immunodeficient) mice suggest that they are not superior to nude mice for establishment of serially transplantable prostatic carcinoma xenografts. CONCLUSIONS: A proportion of human primary prostatic carcinomas can be grown as xenografts. Four new serially transplantable xenografts (CWR21, CWR31, CWR91, and CWR22) are currently propagated in our laboratory, a resource that was not previously available. IMPLICATIONS: Our experience suggests that the most important factor in serial transplantation is the collaboration of urologists and pathologists in expediting placement of the tumor in cold saline, examination of the frozen section, and transplantation.

Chromosome analysis of hepatocellular carcinoma 7777 and correlation with alpha-fetoprotein production.

The Morris hepatocellular carcinoma 7777 is productive of extraordinarily high levels of the oncofetal protein, alpha-fetoprotein. Its chromosome composition was examined in detail since it represents the only near-diploid tumor of such productivity and has been reported to demonstrate a single unusual chromosomal alteration. The major finding is the presence of a submetacentric marker chromosome, composed of a No. 7 chromosome and a short arm that demonstrated a poorly defined banding pattern on Giemsa staining. This marker is unique to 7777 and is of particular interest in view of recent reports of an association between such unbanded chromosome arms and supraproduction of cell products.

Cytogenetic, flow cytometric, and ultrastructural studies of twenty-nine nonfamilial human renal carcinomas.

Multifactorial analysis, including cytogenetic studies, flow cytometry, and light and electron microscopic evaluation, was performed on 29 primary renal cell carcinomas and short-term cultures derived from them. Eleven of the 21 cases that yielded cytogenetic results demonstrated clonal chromosomal aberrations which included trisomy 7 in 8 cases, loss of the Y chromosome in 7, trisomy 12 in 2, and 16q- in 1. Flow cytometry showed that there was preferential growth of near-diploid populations and loss of aneuploid clones in culture with standard media. The ultrastructural features of both the primary and cultured tumors were remarkably similar. They included cytoplasmic vacuolization, reticulated dense nucleoli, and cell surface microvilli. Thus, morphological evidence supported the epithelial and, specifically, the renal tubular origin of the cultured cells. The development of chromosomal abnormalities seemed linked to advanced tumor stage, but the number of such cases was too small to analyze for statistical significance. No other correlations could be made between karyotypic change, DNA analysis, tumor histology, grade, and stage at this point in the patient follow-up.

Irradiation-induced marker chromosomes in a metastasizing murine tumor.

We have used irradiation to induce marker chromosome formation in a metastasizing murine tumor with a stable karyotype. The induced recombinant chromosomes then served to determine whether metastases were of clonal or multicellular origin. Cumulative data were obtained from four series of experiments on spontaneous metastases originating from tumors grown from irradiated cells; 20 of these metastases expressed unique chromosomal alterations consistent with a clonal origin. The majority of metastasis-derived cell populations remain stable with respect to their marker chromosomes in culture as well as in successive animal transplantation. In several instances, however, chromosomal instability was sufficient to obscure the cellular origins of individual metastases. A few metastases showed mixed chromosomal patterns initially that were consistent with multicellular origin, but repeat examinations have revealed a chromosomal instability which persisted during propagation in culture. The frequency of chromosomal recombinants in metastases from the combined series was sufficient to suggest biological and statistical significance. The morphology of recombinants was not associated with radiation dose but appeared as an apparently random response of the tumor population in different experiments. Analysis of chromosomal markers by banding techniques was performed to determine if specific chromosomes or chromosomal sites were associated with tumor cells from metastatic foci (a host-selected subpopulation with a metastatic phenotype). Our results did not reveal preferential involvement of whole chromosomes or intrachromosomal sites in recombinant formation.

Development and progression of karyotypic variability in melanoma K1735 following X-irradiation.

Chromosomal aberrations are often assumed to be deleterious to cells. However, we have found that many metastases are populated by cells with chromosomal recombinants induced by radiation of the original tumor population. The tumor, K-1735-M2, was already capable of metastasis so that the recombinant chromosomes were not necessary for this property of the tumor. Stable recombinants, like other aberrant forms, could be disadvantageous or, alternatively, could confer selective advantage to some tumor cells. We investigated these possibilities by irradiating the parental tumor line and examining the formation and persistence of chromosomal markers in cell culture and in s.c. tumors. The karyotype of the K-1735-M2 parental tumor is composed entirely of telocentric chromosomes, and recombinant forms are relatively easy to recognize. Unstable forms of chromosome damage were lost rapidly. The frequency of stable recombinants after two weeks in culture was higher than that in tumors growing in primary inoculation sites. In contrast, secondary (spontaneous metastatic) foci showed a far greater frequency of chromosomal markers, suggesting a positive association between markers and acquisition of properties benefiting growth and metastasis.

Sequential analysis of transplantable hepatocellular carcinomas.

In a previous study multiple characteristics of chemically induced primary hepatocellular carcinomas were described and examined during the initial transplant generations. The present communication reports on these characteristics in subsequent transplant generations followed over a 2-year period. In almost all instances the growth rate, morphology, chromosome composition, and plasma protein and alpha-fetoprotein synthesis of individual tumors have remained relatively constant. However, one spontaneous subline of a diploid tumor demonstrated a sudden extensive rearrangement of its chromosomes simultaneous with a significant acceleration of growth rate. Despite karyotypic evolution, it retained the functional characteristics of diploid tumors, producing no plasma protein or alpha-fetoprotein.

Molecular cloning and chromosomal mapping of a human galactoside-binding protein.

A human galactoside-binding protein with an Mr of 31,000 was cloned from the human HT-1080 fibrosarcoma complementary DNA library. A partial complementary DNA clone containing the complete coding region was characterized and the deduced sequence encodes a polypeptide of 242 amino acids with the characteristics of a carbohydrate-binding protein. The gene coding for the human galactoside-binding protein was mapped to the chromosomal band 1p13. The deduced amino acid sequence of the human galactoside-binding protein revealed 95 residues at the amino terminus, homologous to the predicted amino acid sequence of the second exon of the human L-myc gene.

Allelic loss in locally metastatic, multisampled prostate cancer.

In order to determine whether retention or loss of potential tumor suppressor loci that map to 8p, 10q, or 16q reflect genetic relationships among prostatic intraepithelial neoplasias (PINs), multicentric primary prostatic cancers, and regional lymph node metastases or are associated with the metastatic phenotype, we analyzed 19 cases of locally metastatic prostate carcinoma (stage D1) utilizing polymerase chain reaction techniques. In each case, tissue samples from metastatic tumor, the (dominant) primary tumor, and nonneoplastic prostatic tissue were examined. In selected cases, allelic loss in additional tumor foci, separate from the dominant tumor nodule, and areas of PIN were examined. Allelic loss of sequences on 8p, 10q, and 16q were observed in 20-29% of PINs, 18-42% of primary tumors, and 8-25% of metastatic tumors. Discrepancies in sequence dosage between histological components were most pronounced for 8p sequences, especially between the dominant tumor nodule and metastatic deposits in cases in which > or = 3 separate tumor foci/gland were identified. These results suggest that putative premalignant lesions, moderately or poorly differentiated, geographically separate primary tumor foci, and metastases within morphologically "complex" prostates (those with > or = 3 foci/gland) are likely to be more discordant for sequence dosage at 8p than those within "simpler" glands (< 3 foci/gland). Also, our results suggest that lymph node metastases may be genetically related to either the dominant or additional primary tumor foci in more complex prostates and that accumulation of genetic aberration may differ in primary and metastatic lesions.

Evolution of methotrexate resistance of human acute lymphoblastic leukemia cells in vitro.

A human acute lymphoblastic T-cell line, MOLT-3, was fed with Roswell Park Memorial Institute Medium 1640 supplemented with 10% fetal bovine serum and antibiotics which contained increasing concentrations of methotrexate (MTX). The development of drug resistance was associated initially with progressive decrease in MTX transport. When the cells became 200-fold resistant, a rise in the dihydrofolate reductase was noted which was short-lived in the absence of the drug. A 10,000-fold increase in MTX resistance was accompanied, in addition to further decrease in MTX transport, by a 10-fold increase in the dihydrofolate reductase activity. While the purely transport-related resistant cell lines had a collateral sensitivity to lipid-soluble antifols, the sublines which had both transport- and enzyme-related MTX resistance contained a subpopulation highly resistant to these antifols. Chromosome analysis of the subline with increased dihydrofolate reductase activity showed an expanded abnormally banded region in chromosome 5.

Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10.

Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 mM) and floating transfers in low calcium (MCF-10F), or by trypsin-Versene passages in the customary (normal) calcium levels, 1.05 mM (MCF-10A). Both sublines have been maintained as separate entities after 2.3 years (849 days) in vitro and at present have been in culture for longer than 4 years. MCF-10 has the characteristics of normal breast epithelium by the following criteria: (a) lack of tumorigenicity in nude mice; (b) three-dimensional growth in collagen; (c) growth in culture that is controlled by hormones and growth factors; (d) lack of anchorage-independent growth; and (e) dome formation in confluent cultures. Cytogenetic analysis prior to immortalization showed normal diploid cells; although later passages showed minimal rearrangement and near-diploidy, the immortal cells were not karyotypically normal. The emergence of an immortal culture in normal calcium media was not an inherent characteristic of the original tissue from which MCF-10 was derived since reactivated cryo-preserved cells from cultures grown for 0.3 and 1.2 years in low calcium were incapable of sustained growth in normal calcium.

The biology of breast cancer at the cellular level.

Two properties seem fundamental to cancer; heterogeneity and progression (Foulds (1975) Academic Press, New York; Heppner et al. (1979) Commentaries on Research in Breast Disease, Vol. 1 (Bulbrook, R. and Taylor, D.J., eds.), pp. 177-191, Plenum Press, New York). Relatively little is understood about the premalignant stages of human breast disease in vivo. When the disease manifests as invasive carcinoma, its behavior exhibits great diversity, sometimes metastasizing rapidly, while in other cases 10-30 years pass before metastases proliferate. Here we review various aspects of breast cancer in vivo and consider how they predict properties of breast cancer found in culture. All of the experiments are consistent with the hypothesis proposed by Nowell (1976) Science 194, 23-28, that a fundamental aspect of malignancy is an increased genetic instability and that many of the cells within tumors are nonviable results of genetic instability. We suggest that most of the viable cells within primary breast carcinomas are diploid and are not yet capable of aspects of metastatic spread. What these cells have attained is an increased propensity for genetic instability which enables them to generate randomly aneuploid but frequently lethal genetic configurations. Occasionally one of these altered genomes is associated with the ability to proliferate at a metastatic site. This hypothesis implies that metastases from various patients may have arisen by divergent pathways and may also be divergent in many other aspects of their physiology, unrelated to malignancy. Such extreme heterogeneity may hamper attempts to understand fundamental aspects of malignancy. Hence we suggest that the less anaplastic and less divergent diploid cells within the primary carcinomas might be an important resource to gain insights into the critical alterations that are responsible for initiating frankly malignant behavior.

HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas.

PURPOSE: The HER-2/neu gene codes for a membrane receptor protein that is homologous, but distinct from the epidermal growth factor receptor. This investigation was performed to validate fluorescence in situ hybridization (FISH) as a sensitive and specific method for assessing HER-2/neu gene amplification in archival tissue and to test whether this alteration is associated with poor prognosis. MATERIALS AND METHODS: HER-2/neu gene amplification was determined by FISH in 140 archival breast cancers, previously characterized for gene amplification by Southern hybridization or dot-blot hybridization, and for gene expression by Northern hybridization, Western immunoblot, or immunohistochemistry. A separate cohort of 324 node-negative breast cancers was assessed for amplification by FISH to determine the utility of HER-2/neu gene amplification. RESULTS: Relative to solid-matrix blotting procedures, FISH analysis of HER-2/neu gene amplification showed a sensitivity of 98% and a specificity of 100% in 140 breast cancers. Among patients treated by surgery only, the relative risks (relative hazard) of early recurrence (recurrent disease within 24 months of diagnosis), recurrent disease (at any time), and disease-related death were statistically significantly associated with amplification. The prognostic information contributed by HER-2/neu amplification was independent of the other markers studied. CONCLUSION: FISH was an alternative technique for determining gene amplification and had some distinct advantages over Southern hybridization. Our results demonstrate that HER-2/neu gene amplification in the absence of adjuvant therapy is an independent predictor of poor clinical outcome and is a stronger discriminant than tumor size. Women with small tumors that had gene amplification were at increased risk of recurrence and disease-related death.

Growth of granulopoietic bone marrow cells of RF mice.

Mice of the RF strain are unusually susceptible to the development of leukemia, both spontaneously and after whole body irradiation. Cell culture and transplantation techniques were used to study the proliferative capacity of hematopoietic tissues of these mice. The growth characteristics of granulopoietic cells of young, non-irradiated RF mice were compared with those from a leukemia-resistant strain, C57BL/6J (B6). Marrow from RF mice showed a substantial reduction in the number of colony-forming units in culture (CFUC). The numbers of marrow cells capable of forming colonies in the spleen of lethally irradiated syngeneic mice (CFUS) were also reduced. The spleens from non-irradiated RF mice were almost twice the size of those from B6 mice. Despite these differences the numbers of circulating peripheral leukocytes and percent of polymorphonuclear leukocytes were similar in the two strains. Bone marrow from RF mice may be analogous to that of certain human "preleukemic" states in which alterations in marrow cells are demonstrable in culture.

NM1 keratinocyte line is cytogenetically and biologically stable and exhibits a unique structural protein.

We have previously described a human keratinocyte line, NM1, which had been carried for more than 400 doublings, was trisomic for chromosome 8, and appeared to make a number of structural proteins characteristic of keratinocytes. This line has now been carried for more than 800 doublings and grows with the same vigor. It reaches confluence in 7 to 10 days and can be grown without a feeder layer for more than 15 passages. Its karyotype has remained 47,XY, +8. The current NM1 cells make readily detectable amounts of 67 kd and 48 kd keratins, and it has been established that the previously poorly resolved 58 kd band actually consists of 58 kd and 59 kd bands. We have also found that the apparent 56 kd band consists of the 56 kd and 56.5 kd bands. A unique basic polypeptide precursor of the cornified envelope has been discovered in the NM1 line. Although similar in charge to one in normal cells it is lower in molecular weight.