CD1A-positive cells and HSP60 (HSPD1) levels in keratoacanthoma and squamous cell carcinoma

CD1a is involved in presentation to the immune system of lipid antigen derived from tumor cells with subsequent T cell activation. Hsp60 is a molecular chaperone implicated in carcinogenesis by, for instance, modulating the immune reaction against the tumor. We have previously postulated a synergism between CD1a and Hsp60 as a key factor in the activation of an effective antitumor immune response in squamous epithelia. Keratoacantomas (KAs) are benign tumors that however can transform into squamous cell carcinomas (SCCs), but the reasons for this malignization are unknown. In a previous study, we found that CD1a-positive cells are significantly more numerous in KA than in SCC. In this study, we analyzed a series of KAs and SCCs by immunohistochemistry for CD1a and Hsp60. Our results show that the levels of both are significantly lower in KA than in SCC and support the hypothesis that KA may evolve towards SCC if there is a failure of the local modulation of the antitumor immune response. The data also show that immunohistochemistry for CD1a and Hsp60 can be of help in differential diagnosis between KAs and well-differentiated forms of SCC.     

IMP-3 expression in keratoacanthomas and squamous cell carcinomas of the skin: an immunohistochemical study

The protein insulin-like growth factor II mRNA binding protein 3 (IMP-3) is an important factor for cell migration and adhesion in malignancies. Recent studies have shown a remarkable overexpression of IMP-3 in different human malignant neoplasms and also revealed it as an important prognostic marker in some tumor entities. The purpose of this study is to compare IMP-3 immunostaining in cutaneous squamous cell tumors and determine whether IMP-3 can aid in the differential diagnosis of these lesions. To our knowledge, IMP-3 expression has not been investigated in skin squamous cell proliferations thus far. Immunohi-stochemical staining for IMP-3 was performed on slides organized by samples from 67 patients, 34 with keratoacanthoma (KA) and 33 with primary cutaneous squamous cell carcinoma (SCC) (16 invasive and 17 in situ). Seventyfour percent of KAs (25/34) were negative for IMP-3 staining, while 57% of SCCs (19/33) were positive for IMP-3 staining. The percentage of IMP-3 positive cells increased significantly in the invasive SCC group (P=0.0111), and particularly in the SCC in situ group (P=0.0021) with respect to the KA group. IMP-3 intensity staining was significantly higher in invasive SCCs (P=0.0213), and particularly in SCCs in situ (P=0.008) with respect to KA. Our data show that IMP-3 expression is different in keratoacanthoma with respect to squamous cell carcinoma. IMP-3 assessment and staining pattern, together with a careful histological study, can be useful in the differential diagnosis between KA e SCC.Key words: IGF-II mRNA-binding protein-3 (IMP-3), keratoacanthoma, squamous cell carcinoma.     

The multi-step process of human skin carcinogenesis: a role for p53, cyclin D1, hTERT, p16, and TSP-1

As proposed by Hanahan and Weinberg (2000. Cell 100, 57-70) carcinogenesis requires crucial events such as (i) genomic instability, (ii) cell cycle deregulation, (iii) induction of a telomere length maintenance mechanism, and (iv) an angiogenic switch. By comparing the expression of p53, cyclin D1, p16, hTERT, and TSP-1 in spontaneously regressing keratoacanthoma (KA) as a paradigm of early neoplasia, with malignant invasive cutaneous squamous cell carcinoma (SCC) as a paradigm of advanced tumour development, we are now able to assign the changes in the expression of these proteins to specific stages and allocate them to defined roles in the multi-step process of skin carcinogenesis. We show that mutational inactivation of the p53 gene, and with that the onset of genomic instability is the earliest event. Individual p53-positive cells are already seen in "normal" skin, and 3/5 actinic keratoses (AKs), 5/22 KAs, and 13/23 SCCs contain p53-positive patches. Cell cycle deregulation was indicated by the overexpression of the cell cycle regulator cyclin D1, as well as by the loss of the cell cycle inhibitor p16. Interestingly, overexpression of cyclin D1 - observed in 80% of KAs and SCCs, respectively - showed a cell cycle-independent function in HaCaT cell transplants on nude mice. Cyclin D1 overexpression was associated with a massive inflammatory response, finally leading to tissue destruction. Loss of the cell cycle inhibitor p16, on the other hand, correlated with SCCs. Thus, it is tempting to suggest that overexpression of cyclin D1 is an early change that in addition to growth stimulation leads to an altered epithelial-mesenchymal interaction, while functional p16 is able to control this deregulated growth and needs to be eliminated for malignant progression. Another requirement for uncontrolled growth is the inhibition of telomere erosion by up-regulating telomerase activity. As measured by hTERT protein expression, all of the KAs and SCCs studied were positive, with a similar distribution of the protein in both groups and an expression pattern resembling that of normal epidermis. Thus, telomerase may not need to be increased significantly in skin carcinomas. Finally, we show that the angiogenesis inhibitor TSP-1 is strongly expressed in most KAs, and mainly by the tumour cells, while in SCCs the generally weak expression is restricted to the tumour-stroma. Furthermore, we provide evidence that the loss of a copy of chromosome 15 is responsible for reduced TSP-1 expression and thereby this aberration contributes to tumour vascularisation (i.e. the angiogenic switch) required for malignant growth.     

Dual-parameter flow cytometric analysis coupling the measurements of forward-angle light scatter and DNA content of archival ovarian carcinomas of low malignant potential

Paraffin-embedded archival specimens from 45 cases of ovarian carcinoma of low malignant potential (OCLMP) were analyzed by flow cytometry (FCM) using propidium iodide (PI) staining. Since single-parameter FCM analysis is often deficient in the resolution of subtle near-diploid DNA-aneuploid populations, forward-angle light scatter (FALS) was measured as a second parameter. DNA aneuploidy was identified in 15 cases (33%). In 7 of those 15 cases, aneuploidy was resolved with single-parameter FCM; in the remaining 8 cases, DNA aneuploidy was resolved only following dual-parameter analysis coupling DNA content and FALS. In all 15 cases, a single near-diploid aneuploid population was observed (mean DNA index = 1.2); there were no tetraploid aneuploid cases. The proliferative activity for all 45 cases studied ranged from 1.0% to 8.9%, with a mean of 3.5%. No difference in mean proliferative activity was observed between the aneuploid and diploid tumors (P greater than .05). To exclude the possibility that PI staining artifacts caused the observed aneuploidy, five of the eight cases shown to be aneuploid by dual-parameter analysis were further studied using an alternate DNA-binding dye, DAPI, yielding similar results. To exclude the possibility that contaminating stromal and/or inflammatory cells caused the observed aneuploidy, samples from a subset of the dual-parameter cases were sorted, revealing the aneuploid populations to be composed primarily of tumor nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unbiased Approach for Virus Detection in Skin Lesions

To assess presence of virus DNA in skin lesions, swab samples from 82 squamous cell carcinomas of the skin (SCCs), 60 actinic keratoses (AKs), paraffin-embedded biopsies from 28 SCCs and 72 kerathoacanthomas (KAs) and fresh-frozen biopsies from 92 KAs, 85 SCCs and 92 AKs were analyzed by high throughput sequencing (HTS) using 454 or Ion Torrent technology. We found total of 4,284 viral reads, out of which 4,168 were Human Papillomavirus (HPV)-related, belonging to 15 known (HPV8, HPV12, HPV20, HPV36, HPV38, HPV45, HPV57, HPV59, HPV104, HPV105, HPV107, HPV109, HPV124, HPV138, HPV147), four previously described putative (HPV 915 F 06 007 FD1, FA73, FA101, SE42) and two putatively new HPV types (SE46, SE47). SE42 was cloned, sequenced, designated as HPV155 and found to have 76% similarity to the most closely related known HPV type. In conclusion, an unbiased approach for viral DNA detection in skin tumors has found that, although some new putative HPVs were found, known HPV types constituted most of the viral DNA.     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

NASA/American Cancer Society High-Resolution Flow Cytometry Project-I

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. METHODS: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. RESULTS: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 +/- 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. CONCLUSIONS: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

Flow cytometric determination of DNA content in malignant and benign bone tumours

The nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S- and G2M-phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p less than 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.     

Flow cytometric DNA measurements in human thyroid tumors

By means of flow cytometry (FCM), DNA distribution pattern and the fraction of cells in the various phases of the cell cycle were studied in 52 samples of normal thyroid tissues, follicular adenomas, follicular carcinomas, medullary carcinoma and fibrosarcomas. In the normal thyroid tissues and follicular adenomas DNA diploid cell populations only were found. Among 20 follicular carcinomas in 13 cases (65%) together with the DNA diploid cells, DNA aneuploid cell lines were also observed. S-phase fraction in follicular adenomas is higher than in the normal thyroid tissues and lower than those in thyroid carcinomas. The percentage of S-phase cells in DNA aneuploid populations is significantly higher (S = 19 +/- 9.3%) than in the diploid cell lines (S = 3.7 +/- 2.6%). DNA aneuploid cell populations were predominantly observed in carcinomas with a high degree of morphological anaplasia.     

Double labelling to obtain S phase subpopulations: application to determine cell kinetics of diploid cells in an aneuploid tumour

Abstract. We studied the cell kinetics of the murine mammary carcinoma MCa-K using iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) given at different times as independently detectable labels of S phase cells. The presence of IdUrd and CldUrd, and the amount of DNA were measured by three-colour flow cytometry making it possible to define three subpopulations within S phase and to measure the progression through the cell cycle during the time following labelling. In DNA histograms of these subpopulations, the diploid and aneuploid cells (which had a DNA index of 1.7) are essentially completely separated. From appropriate combinations of cells labelled with IdUrd only, CldUrd only, or both, it was possible to construct separate DNA distributions for the labelled diploid and aneuploid cells at the times of administration of each label. The kinetics of the diploid and aneuploid cells could be calculated for individual tumours from these two time points without having to make corrections for the presence of the second population. The diploid and aneuploid populations had indistinguishable S and G₂+ M phase durations, T_S and T_G2+M, of about 9 and 2h; however, the potential doubling time values for the aneuploid and diploid populations were 30.2 and 101.2h respectively.     

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.     

Chromosomal composition of aneuploid clones with different DNA contents in head and neck squamous cell carcinomas as determined by combined flow cytometry and fluorescence in situ hybridization.

Studies with DNA flow cytometry (FCM) have shown that DNA contents of aneuploid tumour clones vary in a wide range. The aim of this study was to analyse whether homologous chromosomal changes exist despite the individual differences that may be of general relevance for the development of gross aneuploidy in squamous cell carcinomas of the head and neck. Fluorescence in situ hybridization (FISH) with 13 centromere-specific DNA probes was applied to 3 diploid and 11 aneuploid tumours with DNA indices ranging between 0.8 and 2.2. Disomic and monosomic cell populations were prevalent findings in DNA-diploid tumours. Polysomies were common in aneuploid tumours. Different degrees of aneusomy for identical chromosomes were recurrent features in aneuploid tumours. FISH signal heterogeneity was identified for all chromosomes. The mean number of aneusomic cell populations identified for DNA-aneuploid tumours ranged between 1.6 for chromosome 17 and 3.1 for chromosome 3. Inconsistencies between FISH and FCM data may indicate that centromere-specific DNA probes identify gains and losses of marker DNA due to complex karyotypic rearrangements rather than absolute changes in chromosome numbers. Overall, there was no evidence of the critical involvement of particular chromosomes in the development of different DNA contents.     

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

A correction required for calculation of DNA ratios in flow cytometric analysis of ploidy

Flow cytometric measurements of nuclear DNA content often involve the calculation of a DNA index, which compares the DNA fluorescence from two different populations. Such DNA indices have been used to classify aneuploid peaks from tumour tissue into different categories relative to normal diploid cells. This report describes a correction based on the channel for unstained particles that is required if DNA index values are to give a true and reproducible indication of relative DNA content.     

Detection of residual aneuploid leukemic cells by "continuous gating".

BACKGROUND: A new method for the detection of residual aneuploid leukemic cells in bone marrow by flow cytometry is described. This method is based on the analysis of FCM derived list-mode-datasets with a new software called "Continuous Gating". The program is able to decrease the detection level of aneuploid tumor cells by analyzing groups of cells with comparable antigen density and scatter properties. METHODS: Aneuploid acute lymphocytic leukemia cells with a known CD34 expression were diluted with diploid bone marrow cells to a concentration of 10, 1, 0.1, 0.05, and 0.01%. Each sample was measured in a FACScan flow cytometer, after staining with CD34 Moab and propidium iodide. Listmode-data were analyzed with the new "Windows"-based "Continuous Gating" software. A gate was set in the DNA parameter, defining the channels in which the aneuploid G0/G1-peak of possible residual tumor-cells should be found. Ten thousand overlapping gates of size 200 x 200 channels (out of 1,023 x 1,023 channels) were set automatically by the program into the side-scatter (SSC)/CD34 dot-plot, calculating the percentage of aneuploid G0/G1-phase cells for every specific gate. RESULTS: The results are plotted in a contour-plot. In dot-plot gates with less than 20 cells, the calculation of the percentage of aneuploid cells was declared invalid and the area in the contour-plot was marked. Detection of residual aneuploid cells, based on a defined expression of CD34 and granularity (SSC), was possible down to a contamination of 0.1%. CONCLUSIONS: The new "Continuous Gating" software can be used for the automated detection of aneuploid leukemic cells, if the density of a certain surface-marker is slightly different from normal cells.     

RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12.

Escherichia coli rnh mutants lacking RNase H activity are capable of recA+-dependent DNA replication in the absence of concomitant protein synthesis (stable DNA replication). In rnh dnaA::Tn10 and rnh delta oriC double mutants in which the dnaA+-dependent initiation of DNA replication at oriC is completely blocked, the recA200 mutation encoding a thermolabile RecA protein renders both colony formation and DNA synthesis of these mutants temperature sensitive. To determine which stage of DNA replication (initiation, elongation, or termination) was blocked, we analyzed populations of these mutant cells incubated at 30 or 42 degrees C in the presence or absence of chloramphenicol (CM) by dual-parameter (DNA-light scatter) flow cytometry. Incubation at 30 degrees C in the presence of CM resulted in cells with a continuum of DNA content up to seven or more chromosome equivalents per cell. The cultures which had been incubated at 42 degrees C in the absence or presence of CM consisted of cells with integral numbers of chromosomes per cell. It is concluded that active RecA protein is required specifically for the initiation of stable DNA replication.     

A strategy combining flow sorting and comparative genomic hybridization for studying genetic aberrations at different stages of colorectal tumorigenesis in ulcerative colitis

BACKGROUND: DNA aneuploidy has been shown to increase the risk of developing dysplasia in ulcerative colitis (UC) and is related to tumorigenesis in the colorectum. Therefore, it is of particular interest to study genetic aberrations behind DNA aneuploidization during colorectal carcinogenesis. We wanted to elucidate further the relationship between mucosal morphology and DNA aberrations in UC. METHODS: DNA flow cytometry was applied to multiple lesions including regenerative, dysplastic, and carcinomatous mucosa from the colectomy specimen of a male patient with long-standing UC. The lesions harbored multiple DNA aneuploid stemlines that were subjected to flow sorting. We analyzed gene alterations by degenerate oligonucleotide primer (DOP; universal primers) polymerase chain reaction (PCR)-based comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) in diploid and aneuploid sorted cells. RESULTS: DOP-PCR-based CGH shows gains and losses that can be verified by FISH. We show that with this approach one can study genetic evolution of distinct DNA diploid and aberrant subpopulations through defined stages of colorectal tumorigenesis. This includes getting information related to tumor heterogeneity that cannot be obtained by CGH with DNA extracted from nonsorted cell populations. Genetic imbalance was also detected in diploid nondysplastic flow-sorted mucosal cells from the same bowel. CONCLUSIONS: Similar gains and losses were found in aneuploid dysplasias and carcinomas at widely separated locations in the same bowel, indicating a common selection pressure in different areas of the same bowel. The common aberrations may be of importance for progression from dysplasia to carcinoma.     

The role of DNA flow and image cytometry in the evaluation of body cavity fluids

In addition to conventional cytomorphologic study, 50 body cavity fluid specimens (benign and malignant) were analyzed by both flow cytometry (FCM) and image cytometry (ICM) in order to evaluate the potential application of these techniques in the diagnosis of malignancy. While 88% of the fluids were similarly classified by FCM and ICM as being either diploid (66%) or nondiploid (22%), with similar DNA index values, nondiploid peaks were identified by ICM alone in 12% of the fluids. Aneuploid populations were present in 92% of the cytologically positive fluids and in 15% of the cytologically negative fluids. All of the latter fluids came from patients who clinically had tumors involving the respective body cavities. These results show that (1) both FCM and ICM are useful adjuvant techniques in the evaluation of body cavity fluid specimens and (2) ICM is superior to FCM in the identification of small aneuploid subpopulations. The identification of aneuploid populations by either method is highly suggestive of malignancy.