Loss of sequences on the short arm of chromosome 17 is a late event in squamous carcinoma of the cervix.

AIMS: To determine by fluorescence in situ hybridisation (FISH) whether deletion of D17S34, a subtelomeric probe for 17p, occurs in invasive squamous carcinoma of the cervix, and to determine the extent of such loss by analysis of the p53 and HER2/NEU genes. METHODS: Fourteen invasive squamous cell carcinomas of the cervix were investigated by FISH for D17S34, p53, and HER2/NEU. Dual hybridisation of each probe with the chromosome 17 alpha satellite (D17Z1) probe was performed on paraffin wax embedded sections, and the fluorescence ratios of the paired signals were determined. Broad spectrum human papillomavirus (HPV) typing by ISH and GP5+/6+ polymerase chain reaction was also performed. RESULTS: Twelve tumours were HPV positive, nine with HPV-16, and one each with types 18, 31, and 39. Loss of D17S34 was identified in four tumours, one of which was HPV negative. In one tumour, D17S34 loss was accompanied by loss of p53 only, suggesting that deletion was limited to the p arm. A second tumour showed simultaneous losses of all probes, indicative of whole chromosome 17 loss during tumour growth. The two remaining specimens showed loss of D17S34 only, diffuse in one, and localised within the tumour in the other. Aberrations of p53 or HER2/NEU were not seen independently of D17S34 loss, and loss did not correlate with HPV presence or type. CONCLUSIONS: These data show that D17S34 loss is prevalent, marking 28% of the invasive squamous carcinomas in this study. The observed intratumoral heterogeneity indicates that, at least in some cases, this loss occurs after invasion and is therefore a late event in the path of cervical carcinogenesis.

Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells.

The MCF10 series of cell lines was derived from benign breast tissue from a woman with fibrocystic disease. The MCF10 human breast epithelial model system consists of mortal MCF10M and MCF10MS (mortal cells grown in serum-free and serum-containing media, respectively), immortalized but otherwise normal MCF10F and MCF10A lines (free-floating versus growth as attached cells), transformed MCF10AneoT cells transfected with T24 Ha-ras, and premalignant MCF10AT cells with potential for neoplastic progression. The MCF10AT, derived from xenograft-passaged MCF10-AneoT cells, generates carcinomas in approximately 25% of xenografts. We now report the derivation of fully malignant MCF10CA1 lines that complete the spectrum of progression from relatively normal breast epithelial cells to breast cancer cells capable of metastasis. MCF10CA1 lines display histologic variations ranging from undifferentiated carcinomas, sometimes with focal squamous differentiation, to well-differentiated adenocarcinomas. At least two metastasize to the lung following injection of cells into the tail vein; one line grows very rapidly in the lung, with animals moribund within 4 weeks, whereas the other requires 15 weeks to reach the same endpoint. In addition to variations in efficiency of tumor production, the MCF10CA1 lines show differences in morphology in culture, anchorage-independent growth, karyotype, and immunocytochemistry profiles. The MCF10 model provides a unique tool for the investigation of molecular changes during progression of human breast neoplasia and the generation of tumor heterogeneity on a common genetic background.

Significance of p53 mutations in patients with chronic lymphocytic leukemia: a sequential study of 30 patients.

BACKGROUND: In patients with B-cell chronic lymphocytic leukemia (CLL), considerable disease heterogeneity within clinical stages necessitates the search for relevant prognostic indicators, particularly those that may help to determine the need for early therapeutic intervention. In the current study, the authors investigated the role of p53 mutations and chromosomal abnormalities in 30 patients with CLL. METHODS: Thirty patients were screened for p53 mutations. Half of the group had aggressive disease characterized by leucocytosis, lymph node enlargement, organomegaly, and shortened tumor doubling time. Because 95% of p53 mutations reside in "hot-spot" regions of exons 5-9 of the p53 gene, the authors sequenced these exons completely for mutation detection. RESULTS: Sequence analysis identified p53 mutations in 14 of 30 patients that were distributed equally among patients with aggressive disease and nonaggressive disease. There were six mutations in exon 7, five mutations in exon 5, and one mutation each in exons 6 and 8. Five of 15 patients with clinically aggressive disease had mutations in exon 7. Only one patient with nonaggressive disease had an exon 7 mutation. Abnormal cytogenetics were present in 22 of 30 patients (73%). Most patients with the p53 mutation (13 of 14 patients; 93%) displayed abnormal cytogenetics. Twelve of 15 patients with aggressive disease and 9 of 15 patients with average disease exhibited abnormal karyotypes. CONCLUSIONS: The presence of p53 mutations did not predict clinical behavior or disease outcome, although the frequency of mutations appears to be higher than reported previously. In this study, mutations of exon 7 (5 of 6 patients) occurred in patients with clinically aggressive disease. The significance of this observation warrants further examination.

Molecular approaches to identification of tissue contamination in surgical pathology sections.

The finding of possibly contaminant tissues or cells in surgical or cytology case material can be a challenging problem in diagnostic anatomical pathology samples. The reported rates of occurrence have ranged from 0 to 8.8% (including prospective and retrospective cases). A diagnostically dissimilar tissue fragment, whether contiguous with other tissue or among other fragments within a paraffin section, and which is not incompatible with the case tissue, often requires a rigorous investigation to confirm or deny its relevance to the case. Fluorescence in situ hybridization using dual red and green DNA probes to regions of the X and Y chromosomes, respectively, were used in one case where the potential contaminant was suspected to have originated from a male patient. The putative contaminant tissue fragment was confirmed as male, with cells having one X and one Y chromosome, unlike the other tissue fragments on the slide with two X chromosomes. In a second case, DNA polymorphisms were used to compare allelic patterns that were informative not only in proving the extraneous tissue as a contaminant, but in addition, could be used to trace the latter to its original tissue source. The molecular tools of fluorescence in situ hybridization in sex-mismatched cases and of DNA microsatellite probes that are applicable to paraffin sections can provide definitive identifiers of tissues and individual cells. They are important adjuncts to histology for the anatomical pathologist when faced with the diagnostic problems of tissue contamination encountered in routine practice.

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.

Chromosomal aberrations identified in culture of squamous carcinomas are confirmed by fluorescence in situ hybridisation.

AIMS: Chromosomal aberrations in tumour cells are often not discernable by direct analysis. Although cell culture allows qualitative analysis of the karyotype, potential selection and evolution during growth in vitro may yield misleading data. To determine whether aberrations observed in vitro are representative of the original lesion, chromosomal aberrations found after prolonged growth in vitro of two squamous cell carcinomas of the head and neck (SSCHN) were evaluated with fluorescence in situ hybridisation (FISH) on the original tumour nuclei. METHODS: Specific karyotypic aberrations identified in cultures of two squamous cell carcinomas were targets for FISH analysis on tumour sections. Chromosome painting mixtures were selected based on in vitro karyotypic data. FISH was performed on cultured interphase and metaphase cells, and on histological sections from the original tumours. RESULTS: The 9cen and 17cen probes yielded FISH signals consistent with the aneusomies predicted for the respective chromosomes from the culture karyotypes. Whole chromosome 9 paint confirmed the prior existence in the tumours of i(9p) and i(9q), although only the latter hybridised with the 9cen probe. FISH data also supported in vivo representation of the diploid and tetraploid tumour subclones observed in cultures. In tumour HFH-SCC-8a, FISH results were generally concordant between cultured interphase and metaphase cells and the histological sections, and improved the interpretation of marker chromosomes identified in culture. CONCLUSION: The karyotypes obtained in these cases after prolonged passage in culture were consistent with the genetic alterations in the original tumours.

Research potential of a unique xenograft model of human proliferative breast disease.

A workshop on the 'Research potential of a unique xenograft model of human proliferative breast disease' was held at the Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, in November of 1998. The accumulated information and current experimental findings on the MCF10AT model of preneoplastic, proliferative breast disease were reviewed. Discussions focused on the relevance of the model to clinical breast cancer and on the most profitable lines of further research to strengthen its utility.

A new BRCA1 mutation in a Filipino woman with a family history of breast and ovarian cancer.

Mutation of the BRCA1 gene in well-defined breast cancer families has been associated with an 87% lifetime risk for breast cancer and a 44% risk for ovarian cancer. Recent data indicate that the risk associated with these mutations is considerably lower, although still far greater than the risk for disease in the rest of the population. Approximately 81% of the mutations that have been identified have been frameshift (71%) or nonsense (10%) mutations, and either may result in a truncated protein. The protein truncation test (PTT) is often used to screen patients at high risk, because sequencing of this large (100 kb) gene with its 22 coding exons is an arduous task. The PTT was used to analyze genomic DNA and RNA from the peripheral blood of a 31-year-old Filipino woman with a poorly differentiated, stage 2A breast carcinoma and a family history of breast-ovarian cancer. PTT identified the wild-type protein fragment and an additional truncated protein fragment in the patient's sample. Subsequent direct sequencing of the appropriate coding region revealed a point mutation in exon 11 at nucleotide 2178, resulting in a C > T transition that caused a termination (stop codon) in amino acid 687. To our knowledge, this is the first report of mutation of the BRCA1 gene in a Filipino family, and this in-frame stop-codon mutation has not been reported previously.

Trisomy 6 as a primary karyotypic aberration in hematologic disorders.

We identified seven patients with hematologic disorders and trisomy 6 as the sole karyotypic aberration in bone marrow aspirates or unstimulated peripheral blood. Five patients were male and two were female; all were adults with ages ranging from 22 to 74 years. Three of the seven patients presented with manifestations of peripheral cytopenia. Their bone marrows were hypocellular with slight or no dysplastic changes and without an increase in blasts. One of these patients subsequently developed acute myeloid leukemia (AML-M1). The four remaining patients were initially diagnosed with AML--three consistent with French-American-British classification of M1 and M4 in the fourth patient. These results suggest that trisomy 6 is a nonrandom primary numerical anomaly of myeloid disorders. The association of cytopenia and hypoplastic bone marrow with trisomy 6 may constitute a new, distinctive variant among myelodysplastic syndromes.

Direct action of estrogen on sequence of progression of human preneoplastic breast disease.

We have used the MCF10AT xenograft model of human proliferative breast disease to examine the early effects of estradiol exposure on morphological progression of preneoplastic lesions and to define the step(s) in the morphological sequence at which estrogen may act. The effects of estradiol on neoplastic progression of estrogen-receptor-positive MCF10AT cells in the orthotopic site were examined in ovariectomized female nude mice that received subcutaneous administration of implants of 17beta-estradiol or placebo pellets. At 10 weeks, histological analysis of the lesions derived from the estrogen-supplemented group revealed that 92% of lesions displayed histological features of atypical hyperplasia, carcinoma in situ, or invasive carcinoma, and the remaining 8% exhibited histological features of moderate hyperplasia. These highly proliferative lesions are in marked contrast to the control group in which 60% of samples displayed no evidence of hyperplasia. In contrast with control xenografts, estrogen-exposed xenografts demonstrated extensive areas of papillary growth, adenosis-like areas, prominent host inflammatory infiltration, and angiogenesis. Our results suggest that estrogen exerts a growth-promoting effect on benign or premalignant ductal epithelium by enhancing 1) the frequency of lesion formation, 2) the size of lesions, 3) the speed of transformation from normal/mild hyperplasia to those with atypia, 4) the degree of dysplasia, and 5) angiogenesis.

Chromosomal markers: signposts on the road to understanding neoplastic disease.

Classical cytogenetic studies have defined chromosomal attributes of tumors that are useful at present in the diagnosis, classification, and management of leukemic disorders, and in some solid tumors as well. Recent advances in molecular techniques now permit application of some forms of chromosomal analysis to studies in cytopathology. However, to fully understand the present and potential uses of these tumor markers, it is necessary to have some background and perspective on classical cytogenetics.

Assessing genetic markers of tumour progression in the context of intratumour heterogeneity.

This is a report from the Kananaskis working group on quantitative methods in tumour heterogeneity. Tumour progression is currently believed to result from genetic instability and consequent acquisition of new genetic properties in some of the tumour cells. Cross-sectional assessment of genetic markers for human tumours requires quantifiable measures of intratumour heterogeneity for each parameter or characteristic observed; the relevance of heterogeneity to tumour progression can best be ascertained by repeated assessment along a tumour progressional time line. This paper outlines experimental and analytic considerations that, with repeated use, should lead to a better understanding of tumour heterogeneity, and hence, to improvements in patient diagnosis and therapy. Four general principles were agreed upon at the Symposium: (1) the concept of heterogeneity requires a quantifiable definition so that it can be assessed repeatably; (2) the quantification of heterogeneity is necessary so that testable hypotheses may be formulated and checked to determine the degree of support from observed data; (3) it is necessary to consider (a) what is being measured, (b) what is currently measurable, and (c) what should be measured; and (4) the proposal of working models is a useful step that will assist our understanding of the origins and significance of heterogeneity in tumours. The properties of these models should then be studied so that hypotheses may be refined and validated.

Cytogenetic aberrations and DNA ploidy in soft tissue sarcoma. A Southwest Oncology Group Study.

We performed cytogenetic analysis and determined DNA content by flow cytometry (FCM) on freshly disaggregated tumor biopsies from 45 patients with soft tissue sarcomas (STS). Cytogenetically aberrant clones characterized 30 (67%) tumors, with the remaining 15 yielding normal karyotypes with or without nonclonal aberrations. No tumors with multiple unrelated clones were observed. Among the 30 tumors with clonally abnormal karyotypes, 21 (70%) had near-diploid stemlines, six were near-triploid and three were near-tetraploid. Ten of the clonally aberrant tumors contained nonrandom chromosomal translocations characteristic of histologic subtypes. Overrepresentation of chromosomes 7 and 8 were common numerical aberrations. Structural aberrations most often involved chromosomes 1, 7, 9, 12, and 14. Clustering of breaks in 9p resulting in partial loss of the short arm was frequent. Unstable aberrations including rings, dicentrics, large markers, small numbers of double minutes, and telomeric associations were seen in nine tumors. With FCM, 27 (60%) tumors had aneuploid DNA content and 18 (40%) were DNA diploid. Of those 18 DNA diploid tumors, 11 showed clonal karyotypic aberrations. In addition, apparent discrepancies between the results of the cytogenetics and FCM with respect to ploidy pattern were seen in 13 samples; 11 had DNA content in the peritriploid to peritetraploid range but the corresponding karyotype was normal or near-diploid. When the findings of the cytogenetics and DNA content analyses were combined, an abnormal cell population by one or both methods was detected in 38 (84%) tumors. The concurrent application of standard cytogenetics and DNA ploidy by FCM provide complementary information confirming a high incidence of genetic alterations in STS.

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.

New directions in breast cancer research.

Research in breast cancer extends in many directions, stimulated by concerns related to the high incidence of the disease and the relative unpredictability of its clinical course. Examples of work in several directions are presented here arranged by four levels of analysis. 1) Molecular, intracellular events (molecular genetics). Recent identification of genes that predispose to breast cancer, and the isolation of those genes and their protein products, permit investigations of the most critical issues: the roles of these genes in normal development and breast differentiation, and how their alteration permits or contributes to tumor initiation. Thus, we expect that understanding the functions of the genes involved in inherited susceptibility to breast cancer will also be informative for sporadic breast cancers. 2) Cellular biology (cellular models for preneoplastic disease). We examine models of breast cancer development and ask how they help to validate a morphologic sequence for human breast neoplasia and whether they permit investigation of how to modify disease progression. Two useful models, one in transgenic mice and the other using human breast stem cells capable of culture and xenograft growth, are now available. 3) Tissue and organ (the tumor and its local environment). We look at the relationship of the tumor cell population to its local environment (stroma, blood vessels, etc.). This leads naturally to questions of how neighboring tissues and cytokines may modify tumor growth. 4) The individual as an organism and member of a population (hormonal rise and chemoprevention). We address identification of the primarily hormonal risk factors and a possible related mode of cancer prevention.

ras and p53 genes in male breast cancer.

We have studied the roles of Ki-ras oncogene and p53 tumor suppressor gene in a series of 20 cases of male breast cancer and one papilloma of the male breast. Ki-ras was detected in 50-microns sections after digestion with proteinase K and SDS. DNA was amplified by polymerase chain reaction, dot blotted, and mutations were screened with labeled ras mutation-specific oligonucleotides. Wild-type and mutant p53 protein were detected with antibodies CM1 and DO7, using the avidin-biotin-peroxidase method. Two of 17 carcinomas showed Ki-ras mutations, both in codon 12 (gly --> lys and gly --> arg). Five of 20 male breast cancers (25%), including one large intraductal carcinoma, expressed mutant p53 protein. Although the incidence of mutant p53 expression in male breast cancer is similar to that in women, Ki-ras mutations are not significantly increased.

The biological significance of the multidrug resistance gene MRP in inversion 16 leukemias.

Multidrug resistance represents an important mechanism by which leukaemic and solid tumour cells escape cell death after exposure to anthracyclines and other natural products. Acute myeloid leukaemia (AML) associated with the inversion chromosome 16: inv(16)(p13q22) has a favourable prognosis and is known to be chemosensitive. The inversion chromosome is seen in a number of FAB subclasses but is most commonly associated with acute myelomonocytic leukaemia with abnormal eosinophils, M4Eo. It results in the creation of a fusion between the myosin heavy chain gene (MYH11) on the short arm and the gene for a transcription factor, core binding factor beta (CBFB) on the long arm. In a subset of these inv(16) AML patients, inversion also results in loss of the gene for the multidrug resistance protein (MRP) at the short arm breakpoint. This gene maps to 16p13.13, centromeric to the primary short arm breakpoint, separated from MYH11 by a distance of approximately 150kb. Deletion of the MRP gene has been demonstrated by in situ hybridisation, gene dosage studies and by loss of heterozygosity of a flanking microsatellite marker (D16S405). Twenty two patients with inv(16) leukaemia were analysed for deletion of the MRP gene. Deletion of the gene was detected in seven patients, fourteen patients showed retention of the gene and in one case the findings were indeterminate. Clinical data from 13 of these patients were analysed revealing deletion of the MRP gene to be significantly associated with longer time from diagnosis until failure (death or relapse from complete remission) in these patients (p = 0.007). From this work and the growing literature concerning MRP, it appears likely that the deletion of an MRP allele, may favourably affect the biology of inv(16) AML and may have important prognostic implications.