Multiparameter DNA content analysis identifies distinct groups in primary breast cancer.

BACKGROUND: Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique. METHODS: The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox's Regression (CR) analysis. RESULTS: Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76-1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18-1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and 'multiploid tumours' (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (10%) independently associated with poor overall survival (P=0.027, CR). CONCLUSION: Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.

Increased frequency of 20q gain and copy-neutral loss of heterozygosity in mismatch repair proficient familial colorectal carcinomas.

Many hereditary nonpolyposis colorectal cancers (CRCs) cannot be explained by Lynch syndrome. Other high penetrance genetic risk factors are likely to play a role in these mismatch repair (MMR)-proficient CRC families. Because genomic profiles of CRC tend to vary with CRC susceptibility syndromes, our aim is to analyze the genomic profile of MMR-proficient familial CRC to obtain insight into the biological basis of MMR-proficient familial CRC. We studied 30 MMR-proficient familial colorectal carcinomas, from 15 families, for genomic aberrations, including gains, physical losses, and copy-neutral loss of heterozygosity LOH (cnLOH) using SNP array comparative genomic hybridization. In addition, we performed somatic mutation analysis for KRAS, BRAF, PIK3CA and GNAS. The frequency of 20q gain (77%) is remarkably increased when compared with sporadic CRC, suggesting that 20q gain is involved in tumor progression of familial CRC. There is also a significant increase in the frequency of cnLOH and, as a consequence, a reduced frequency of physical loss compared with sporadic CRC. The most frequent aberrations observed included gains of 7p, 7q, 8q, 13q, 20p and 20q as well as physical losses of 17p, 18p and 18q. Most of these changes are also observed in sporadic CRC. Mutations in KRAS were identified in 37% of the MMR-proficient CRCs, and mutations in BRAF were identified in 16%. No mutations were identified in PIK3CA or chromosome 20 candidate gene GNAS. We show that the patterns of chromosomal instability of MMR-proficient familial CRC are clearly distinct from those from sporadic CRC. Both the increased gain on chromosome 20 and the increased levels of cnLOH suggest the presence of yet undiscovered germline defects that can, in part, underlie the cancer risk in these families.

PTEN in colorectal cancer: a report on two Cowden syndrome patients.

Heterozygous germline PTEN mutations cause Cowden syndrome. The risk of colorectal cancer in Cowden patients, however, remains a matter of debate. We describe two patients presenting with colorectal cancer at a young age (28 and 39 years) and dysmorphisms fitting the Cowden spectrum. Heterozygous germline mutations in PTEN were found in both patients. Moreover, analysis of the resected colorectal cancer specimens revealed loss of heterozygosity at the PTEN locus with retention of the mutated alleles, and greatly reduced or absent PTEN expression. Histologically and molecularly, the tumours showed resemblance with sporadic colorectal cancers, although they had prominent fibrotic stroma. Our data indicate that PTEN loss was involved in carcinogenesis in the two patients, supporting that colorectal cancer is part of the Cowden syndrome-spectrum. This is in line with data on sporadic colorectal cancer, mice studies and emerging epidemiological data on Cowden syndrome. Although the exact role of germline PTEN mutations in the carcinogenesis of colorectal cancer remains unclear, we think that Cowden syndrome should be in the differential diagnosis of colorectal cancer certainly in view of the possible prognostic and therapeutic consequences. Prospective follow-up and surveillance of PTEN mutation carriers from the age of 25 to 30 years in a study setting should clarify this issue.

Multiple genetic alterations cause frequent and heterogeneous human histocompatibility leukocyte antigen class I loss in cervical cancer.

The nature and frequency of human histocompatibility leukocyte antigen (HLA) class I loss mechanisms in primary cancers are largely unknown. We used flow cytometry and molecular analyses to concurrently assess allele-specific HLA phenotypes and genotypes in subpopulations from 30 freshly isolated cervical tumor cell suspensions.Tumor-associated HLA class I alterations were present in 90% of the lesions tested, comprising four altered pheno/genotype categories: (a) HLA-A or -B allelic loss (17%), mostly associated with gene mutations; (b) HLA haplotype loss, associated with loss of heterozygosity at 6p (50%). This category included cases with additional loss of a (third) HLA-A or -B allele due to mutation, as well as one case with an HLA class I-negative tumor cell subpopulation, caused by a beta2-microglobulin gene mutation; (c) Total HLA class I antigen loss and retention of heterozygosity (ROH) at 6p (10%); and (d) B locus or HLA-A/B downregulation associated with ROH and/or allelic imbalance at 6p (10%). Normal HLA phenotypes and ROH at 6p were observed in 10% of the cases. One case could not be classified (3%). Altered HLA class I antigen expression occurs in most cervical cancers, is diverse, and is mainly caused by genetic changes. Combined with widespread tumor heterogeneity, these changes have profound implications for natural immunity and T cell-based immunotherapy in cervical cancer.

Targetable gene fusions identified in radioactive iodine refractory advanced thyroid carcinoma.

Objective Gene alterations leading to activation of the MAPK pathway are of interest for targeted therapy in patients with advanced radioactive iodine refractory (RAI-R) thyroid carcinoma. Due to technical reasons gene fusion analysis in RNA isolated from formalin-fixed tumor tissues has till now been limited. The objective of the present study was to identify targetable gene rearrangements in RNA isolated from formalin-fixed RAI-R thyroid carcinomas. Design Retrospective study in 132 patients with RAI-R thyroid carcinoma (59 papillary-, 24 follicular-, 35 Hürthle cell- and 14 anaplastic thyroid carcinoma). Methods Total nucleic acid (undivided DNA and RNA) was isolated from formalin-fixed tissue. Extensive gene fusion analysis was performed in all samples that tested negative for pathogenic BRAF, NRAS, HRAS and KRAS variants. Results Seven targetable gene fusions were identified in the remaining 60 samples without known DNA variants. This includes frequently reported gene fusions such as CCDC6/RET (PTC1), PRKAR1A/RET (PTC2) and ETV6/NTRK3 , and gene fusions that are less common in thyroid cancer (TPM3/NTRK1, EML4/ALK and EML4/NTRK3). Of note, most gene fusions were detected in papillary thyroid carcinoma and MAPK-associated alterations in Hürthle cell carcinomas are rare (2/35). Conclusion Targetable gene fusions were found in 12% of RAI-R thyroid carcinoma without DNA variants and can be effectively identified in formalin-fixed tissue. These gene fusions might provide a preclinical rationale to include specific kinase inhibitors in the treatment regimen for these patients. The latter intends to restore iodine transport and/or take advantage of the direct effect on tumor cell vitality once progressive disease is seen.

T-cell retargeting using bispecific monoclonal antibodies in a rat colon carcinoma model. I. Significant bispecific lysis of syngeneic colon carcinoma CC531 is critically dependent on prolonged preactivation of effector T-lymphocytes by immobilized anti-T-cell receptor antibody.

In order to develop a rat model that reflects human weakly or nonimmunogenic tumor-host relationships and allows investigation of T-cell retargeting with bispecific monoclonal antibodies in vivo, we prepared several mixed hybridomas. One fusion partner was the anti-rat-T-cell receptor (TCR)-framework hybridoma R73 and the others were hybridomas producing antibodies against CC531, a Wag rat colon carcinoma. Stimulation of Wag rat spleen cells with immobilized R73 mAb and rIL-2 yielded predominantly CD8 positive effector T-lymphocytes, which lysed control P815 target cells efficiently in R73-mediated reverse antibody-dependent cellular cytotoxicity (ADCC). The capacity of these effectors to cause significant hybrid antibody-mediated lysis of CC531 emerged several days later, was critically dependent on prolonged stimulation with immobilized R73, and was associated with increased N-alfa-benzyloxycarbonyl-L-lysine thiobenzyl esterase content.

The role of protein kinase C activity in the killing of Chinese hamster ovary cells by ionizing radiation and photodynamic treatment.

In several recent studies it has been shown that protein kinase C (PKC) activity may either potentiate or antagonize cell killing by different cytotoxic agents. These apparently conflicting observations suggest that the effects of PKC activity on cell survival may depend on the different properties of different cell types but do not exclude the possibility that the effects may also depend on the nature of the cytotoxic agent. In this context the effects of PKC activation and PKC inhibition or down-regulation on Chinese hamster ovary (CHO) cell survival after photodynamic treatment and ionizing radiation were studied. It appeared that PKC activation by short-term incubation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) protected CHO cells against ionizing radiation but, in contrast, sensitized the cells to photodynamic treatment. Conversely, inhibition of PKC by H7 and down-regulation of PKC activity by prolonged incubation with TPA sensitized CHO cells to ionizing radiation but protected the cells against photodynamic treatment. These results demonstrate that in one particular cell type PKC activity may have opposite effects on cell survival following cellular damage, depending on the nature of the cytotoxic agent.

Molecular genetic analysis of flow-sorted and microdissected ovarian tumor cells improved detection of loss of heterozygosity.

Study of loss of heterozygosity (LOH) is widely used to identify chromosomal locations of putative tumor suppressor genes. In this type of analysis, DNA extracted from tumor tissue is compared with constitutive DNA from the same patient by the use of polymorphic DNA markers (1). This approach has two intrinsic limitations. First, tumor specimens with a high fraction of nonneoplastic cells have to be excluded from this analysis because LOH in tumor cells may be undetectable, because of the low concentration of tumor DNA. This may lead to a selection bias, which affects the representativity of the results. A second limitation is that the analysis of DNA extracted from homogenized tumor samples may obscure the presence of intratumor genetic heterogeneity.

Simultaneous flow cytometric detection of DNA and cellular protein molecules.

Flow cytometry (FCM) is a sophisticated technique for rapid simultaneous analysis of multiple cellular parameters. The technique is based on the interception of a hydrodynamically focused stream of single cells in suspension by an excitation light beam at high speed. As a result, each cell produces light scattering and fluorescence, proportional to the cell size, shape, structure, and fluorescently labeled cellular components, respectively.

Loss of CDC4/FBXW7 in gastric carcinoma.

BACKGROUND: CDC4/FBXW7, encoding a ubiquitin ligase, maps to 4q32 and has been implicated as a tumor suppressor gene and therapeutic target in many tumor types. Mutations in colonic adenomas, and the frequent losses on 4q described in gastric cancer prompt speculation about the role of CDC4/FBXW7 in gastric carcinogenesis. METHODS: We assessed the role of CDC4/FBXW7 in gastric cancer, through loss of heterozygosity (LOH) and multiplex ligation-dependent probe amplification (MLPA) on 47 flow-sorted gastric carcinomas including early-onset gastric cancers (EOGC) and xenografted conventional gastric carcinomas. Ploidy analysis was carried out on 39 EOGCs and immunohistochemistry of CDC4/FBXW7 and its substrates c-myc, c-jun, Notch and cyclin E was performed on 204 gastric carcinomas using tissue microarrays (TMAs). Sequence analysis of CDC4/FBXW7 was carried out on gastric carcinoma cell lines and xenografts. RESULTS: Loss of heterozygosity of CDC4/FBXW7 occurred in 32% of EOGCs, and correlated with loss of expression in 26%. Loss of expression was frequent in both EOGC and conventional gastric cancers. No CDC4/FBXW7 mutations were found and loss of CDC4/FBXW7 did not correlate with ploidy status. There was a significant correlation between loss of CDC4/FBXW7 expression and upregulation of c-myc. CONCLUSION: Loss of CDC4/FBXW7 appears to play a role in both EOGC and conventional gastric carcinogenesis, and c-myc overexpression is likely to be an important oncogenic consequence of CDC4/FBXW7 loss.

The cytotoxic effect of phenol and ethanol on the chondrosarcoma-derived cell line OUMS-27: an in vitro experiment.

Surgery is considered to be the most effective treatment for cartilaginous tumours. In recent years, a trend has emerged for patients with low-grade tumours to be treated less invasively using curettage followed by various forms of adjuvant therapy. We investigated the potential for phenol to be used as an adjuvant. Using a human chondrosarcoma-derived cartilage-producing cell line OUMS-27 as an in vitro model we studied the cytotoxic effect of phenol and ethanol. Since ethanol is the standard substance used to rinse phenol out of a bone cavity, we included an assessment of ethanol to see whether this was an important secondary factor with respect to cell death. The latter was assessed by flow cytometry. A cytotoxic effect was found for concentrations of phenol of 1.5% and of ethanol of 42.5%. These results may provide a clinical rationale for the use of both phenol and ethanol as adjuvant therapy after intralesional curettage in low-grade central chondrosarcoma and justify further investigation.

High frequency of copy-neutral LOH in MUTYH-associated polyposis carcinomas.

Genetic instability is known to drive colorectal carcinogenesis. Generally, a distinction is made between two types of genetic instability: chromosomal instability (CIN) and microsatellite instability (MIN or MSI). Most CIN tumours are aneuploid, whereas MSI tumours are considered near-diploid. However, for MUTYH-associated polyposis (MAP) the genetic instability involved in the carcinogenesis remains unclear, as near-diploid adenomas, aneuploid adenomas and near-diploid carcinomas have been reported. Remarkably, our analysis of 26 MAP carcinomas, using SNP arrays and flow sorting, showed that these tumours are often near-diploid (52%) and mainly contain chromosomal regions of copy-neutral loss of heterozygosity (LOH) (71%). This is in contrast to sporadic colon cancer, where physical loss is the main characteristic. The percentage of chromosomal gains (24%) is comparable to sporadic colorectal cancers with CIN. Furthermore, we verified our scoring of copy-neutral LOH versus physical loss in MAP carcinomas by two methods: fluorescence in situ hybridization, and LOH analysis using polymorphic markers on carcinoma fractions purified by flow sorting. The results presented in this study suggest that copy-neutral LOH is an important mechanism in the tumorigenesis of MAP.

Improved single laser measurement of two cellular antigens and DNA-ploidy by the combined use of propidium iodide and TO-PRO-3 iodide.

Recently, Frey (Cytometry 17:310-318, 1994) demonstrated that TO-PRO-3 iodide (TP3) can be excited indirectly by a 488 nm laser line through energy transfer by propidium iodide (PI). In the present study, we investigated whether PI-TP3 energy transfer can help to overcome spectral cross talk problems associated with the combined use of fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), and PI. Mixtures of keratin 8/18 FITC-labeled, keratin 8/18-PE-labeled, and unlabeled MCF-7 breast carcinoma cells were prepared and stained for DNA with PI (100 microM). The effect of adding a range of TP3 concentrations (0.001 to 16 microM) to these mixtures was evaluated. The combined use of PI and TP3 was further evaluated using mixtures of unlabeled and p53 FITC-labeled COV362.cl4 ovarian cancer cells and mixtures of unlabeled and p53 FITC-labeled COV362.cl4 cells and peripheral blood lymphocytes (PBL), additionally stained for keratin 8/18 (PE). Finally, a human ovarian ascites tumor specimen was triple-stained for keratin 8/18 (PE), vimentin (FITC) and DNA or keratin 8/18 (PE), PCNA (FITC) and DNA. Addition of TP3 allowed complete correction for spectral cross talk of PE/PI into the green fluorescence detector (FL1). Only minimal (FL1 - %FL2) compensation was required at a TP3 concentration of 2.0 microM in the presence of PI (100 microM). The PI spectral cross talk into the orange fluorescence detector (FL2) was reduced by about 50% using the same photomultiplier (PMT) settings. Although addition of TP3 reduced the signal-to-background ratio by about 30%, the advantage gained through full compensation for spectral cross talk resulted in an improved discrimination of p53-positive and -negative subpopulations in a mixture of human PBL and COV362.cl4 cells. Furthermore, vimentin-negative and PCNA-negative cells were better resolved in a human DNA-aneuploid ovarian ascites tumor after staining the DNA with PI/TP3, rather than with PI alone. We conclude that the addition of TP3 to PI improves the combined measurement by single-laser flow cytometry of DNA-ploidy and antigen expression in heterogenous clinical samples.

Simultaneous measurement of two cellular antigens and DNA using fluorescein-isothiocyanate, R-phycoerythrin, and propidium iodide on a standard FACScan.

Multiparameter flow cytometry is a powerful tool for analyzing the phenotypic, cell kinetic, and ploidy heterogeneity of tumor cell populations. Because of the substantial spectral overlap of propidium iodide (PI) and R-phycoerythrin (PE) fluorescence emission, this combined use of these fluorochromes has been thought not to be feasible on a standard flow cytometer for these kind of studies. Instead of PI, 7-amino-actinomycin D (7-AAD) is used as DNA stain. In this paper however, we show that PI can be used as a DNA stain in combination with fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) on a standard FACScan. Three established ovarian cancer cell lines (IGROV1, NIH: OVCAR-3, and COV362.c14) were used for these experiments. Cells were fixed with 1.0% paraformaldehyde and permeabilized with various concentrations of lysolecithin for the simultaneous detection of surface antigens by monoclonal antibodies MOv18, BMA180 or OV632, intermediate filament antigens (keratin 18 or vimentin), and DNA. A final concentration of 80 micrograms/ml lysolecithin was found to give optimal results. The emission spectrum overlap from PI into the orange fluorescence channel (FL2) used for PE fluorescence detection could be sufficiently compensated up to a photomultiplier tube potential of about 440 Volts (V) required at the FL2 channel. Using the same instrument settings, 5.10 x 10(4) PE equivalents were detectable. Under these conditions, CVs obtained for the DNA histograms ranged from 3.0-4.1. Application of the method on a mixture of activated peripheral blood lymphocytes and ovarian tumor cells resulted in a clear separation of the two populations both by surface and cytoplasmic antigen expression and DNA content.

Distinction between HLA class I-positive and -negative cervical tumor subpopulations by multiparameter DNA flow cytometry.

BACKGROUND: The study of the molecular-genetic basis of heterogeneity of HLA class I expression in solid tumors is hampered by the lack of reliable rapid cell-by-cell isolation techniques. Hence, we studied the applicability of a flow cytometric approach (Corver et al.: Cytometry 2000;39;96-107). METHODS: Cells were isolated from five fresh cervical tumors and simultaneously stained for CD45 or vimentin (fluorescein isothiocyanate fluorescence), Keratin (R-phycoerythrin fluorescence), HLA class I (APC fluorescence), and DNA (propidium iodide fluorescence). A dual-laser flow cytometer was used for fluorescence analysis. Tissue sections from the corresponding tumors were stained for HLA class I antigens, keratin, vimentin, or CD45. RESULTS: Flow cytometry enabled the simultaneous measurement of normal stromal cells (vimentin positive), inflammatory cells (CD45 positive), epithelial cells (keratin positive), and DNA content readily. Normal stromal/inflammatory cells served as intrinsic HLA class I-positive as well as DNA-diploid references. Good DNA histogram quality was obtained (average coefficient of variation < 4%). Intratumor keratin positive subpopulations differing in HLA class I expression as well as DNA content could be clearly identified. Losses of allele-specific HLA class I expression found by immunohistochemistry were also detected by flow cytometry. CONCLUSIONS: We conclude that multiparameter DNA flow cytometry is a powerful tool to study loss of HLA class I expression in human cervical tumors. The method enables flow-sorting of discrete tumor and normal cell subpopulations for further molecular genetic analysis.

Limited loss of nine tumor-associated surface antigenic determinants after tryptic cell dissociation.

Disodium ethylenediaminetetraacetic acid (EDTA) or trypsin/EDTA are frequently used for the dispersion of monolayer cells into single cell suspensions allowing flow cytometric analysis of surface antigenic determinants. A disadvantage of EDTA is the slow action of this agent, whereas trypsin might affect the antigenic determinants under focus. We studied the possible deleterious effect of trypsin on three different ovarian carcinoma cell lines, COV413b, COV362.c14, and NIH:OVCAR-3, on cell surface antigenic determinants by flow cytometry. Either EDTA or trypsin/EDTA was used for detachment and dissociation of monolayer ovarian cancer cell lines, followed by indirect immunofluorescence with a panel of monoclonal antibodies directed against nine different surface antigenic determinants, including six markers directed against widely distributed antigens. Compared to EDTA, trypsin/EDTA resulted in higher total cell yields and rapid detachment and dissociation into single cell suspensions with significantly lower amounts of dead cells detected by both trypan blue and propidium iodide (PI). Large differences in antigen expression were observed for the different cell lines. However, all antigenic determinants tested could still be detected after tryptic proteolysis. Three antigenic determinants were significantly decreased after trypsin/EDTA compared to EDTA detachment. CA 125 was decreased on COV362.c14 and NIH: OVCAR-3 cells, respectively. BMA 180 and ICAM-1 were decreased on COV413b cells. This cell line-dependent decrease might be caused by differences in glycosylation. We conclude that trypsin/EDTA can be used for rapid monolayer cell detachment with high cell yields and limited loss of antigenic determinants tested.

Molecular genetic analysis of flow-sorted ovarian tumour cells: improved detection of loss of heterozygosity.

Detection of loss of heterozygosity (LOH) is usually performed on homogenised tumour specimens. In this type of analysis samples with a low percentage of tumour cells have to be excluded and possible intra-tumour heterogeneity is obscured. In this study we report the application of polymerase chain reaction (PCR)-driven LOH detection with in total 22 microsatellite markers for chromosome 1q, 3p, 3q, 4p, 6p, 6q, 11p, 11q, 17p, 17q, 18p, 18q, Xp and Xq on flow-sorted cells from fresh and paraffin-embedded ovarian tumour tissue. Titration experiments showed that LOH can be detected with as few as 100 cell equivalents of DNA. Clear examples of LOH could be detected in the sorted aneuploid fractions from one unilateral and two bilateral ovarian tumours from three patients. In two samples the sorted fraction was less than 10% of the total sample. The bilateral tumours from the same patient showed loss of identical alleles for one marker (case OV64) and two markers (case OV69), indicative of their monoclonal origin. Multiparameter flow cytometry using two different ovarian tumour markers (MOv18 and BMA180), an anti-cytokeratin monoclonal antibody (MAb) (M9), an anti-vimentin MAb (V9) and a MAb against the panepithelial antigen 17-1A on the fresh ascites cells of the fourth ovarian cancer patient was used to investigate possible intra-tumour heterogeneity. We showed the presence of at least three phenotypically different populations, of which the diploid, keratin-positive, vimentin-negative population showed a similar LOH pattern as the aneuploid population (DNA index = 1.7), indicative of its neoplastic origin. The same LOH pattern was shown in an omentum metastasis from this patient also having the same aneuploid DNA index of 1.7. The sharing of the same LOH pattern by the diploid and aneuploid tumour cell populations suggests that the observed allele loss events occurred before the development of aneuploidy. PCR on flow-sorted cells is thus an important tool to study clonal diversity in tumours.

Allelotype analysis of flow-sorted breast cancer cells demonstrates genetically related diploid and aneuploid subpopulations in primary tumors and lymph node metastases.

Flow cytometric DNA content measurements have demonstrated extensive DNA ploidy heterogeneity in primary breast carcinomas. However, little is known at the molecular level about the clonal relationship between these tumor cell subpopulations, or about the molecular genetic changes associated with aneuploidization. We have used flow cytometric cell sorting to dissect some of this complexity by isolating clonal subpopulations in breast carcinomas for comparative molecular genetic analysis. Clonal subpopulations were isolated from 12 primary breast carcinomas and 5 lymph node metastases from 4 cases based on DNA content and cytokeratin 8/18 labeling. DNA from these clones was screened for allelic imbalances with 92 polymorphic microsatellite markers mapped to 39 different chromosome arms. Diploid and aneuploid populations were concurrently present in 11 out of 12 primary tumors. The DNA ploidy status of primary tumors was identical to that of the related lymph node metastases. Allelic imbalance was present in 10 out of 11 diploid clones (mean, 3.4 +/- 4.2). All allelic imbalances observed in the diploid clones recurred in the cognate aneuploid clones, but were, in the latter, accompanied by additional allelic imbalances at other loci and/or chromosome arms (mean, 10.9 +/- 5.8). In only two of the four metastatic cases did the allelotypes of metastatic clones show small differences relative to their cognate primary tumors. The primary diploid tumor clone recurred in all lymph node metastases. This study indicates that the majority of allelic imbalances in breast carcinomas are established during generation of DNA ploidy diversity. Recurrence of the allelic imbalances in diploid clones in the aneuploid clones suggests linear tumor progression, whereas the simultaneous presence of early diploid and advanced aneuploid clones in both primary and metastatic tumor sites suggests that acquisition of metastatic propensity can be an early event in the genetic progression of breast cancer.

Four-color multiparameter DNA flow cytometric method to study phenotypic intratumor heterogeneity in cervical cancer.

BACKGROUND: Multiparameter DNA flow cytometry using a one-laser bench-top flow cytometer has been restricted to three different colors. The two laser FACSCalibur has recently been introduced, allowing four-color analysis. Therefore, we optimized and extended our three-color method (Corver et al., 1994, Corver et al. 1996) to a four-color analysis of phenotypic intra-tumor heterogeneity using a bench-top flow cytometer. METHODS: First, the effect of a range of different propidium iodide (PI) and TO-PRO-3 iodide (TP3) concentrations on the coefficient of variation (CV) of the DNA histograms was measured using paraformaldehyde-fixed lysolecithin-permeabilized peripheral blood lymphocytes (PBLs) and SiHa and HeLa cervical cancer cells. Second, labeling freshly isolated cervical cancers from solid tumors was optimized with a mixture of anti-keratin antibodies. Third, the FACSCalibur hardware was modified, thereby allowing the simultaneous measurement of allophycocyanin (APC) fluorescence (FL4) in combination with FL3 pulse processing (FL3-W vs. FL3-A). The optimized procedure was then applied to cell suspensions from four different human cervical cancers to study phenotypic intratumor heterogeneity. Cell suspensions were simultaneously stained for DNA (PI, fluorescence) and three cellular antigens: (a) the epithelial cell-adhesion molecule (Ep-CAM; APC fluorescence), (b) keratin (R-phycoerythrin [RPE] fluorescence) to identify the epithelial fraction, and (c) vimentin (fluorescein-isothiocyanate [FITC] fluorescence) to label stromal cells. RESULTS: Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50-100 microM for all cells tested. Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa). Optimal TP3 concentrations were 0.25-2.0 microM. Average CVs were 2. 58% (PBL), 5.16% (HeLa), and 3.96% (SiHa). Inter- or intra-DNA stem line heterogeneity of Ep-CAM expression was observed in the keratin-positive fractions. Vimentin-positive, keratin-negative cells were restricted to the DNA diploid fraction. CONCLUSIONS: PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells. Four-color high-resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules. The FACSCalibur bench-top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories.