Heterogeneity in renal cell carcinoma and its impact no prognosis--a flow cytometric study.

In the process of tumour progression genetic instability is the basis for the evolution of tumour cell clones with various genotypic and phenotypic characteristics causing heterogeneity. Renal cell carcinoma has a long prediagnostic growth period, which increases the probability of clonal evolution. We have studied 200 consecutive renal cell carcinomas, addressing the interrelationship between intratumour heterogeneity and clinicopathological factors. DNA ploidy patterns were analysed in multiple samples from each tumour using flow cytometry and compared with clinical stage, tumour invasion, metastatic rate and survival. Eighty-five of 192 evaluable tumours (44%) were homogeneous concerning DNA ploidy (62% diploid, 38% aneuploid). Among 107 heterogeneous tumours a majority (79%) contained aneuploid as well as diploid cell clones. Homogeneously diploid tumours had a lower incidence of local tumour spread compared with tumours with aneuploid cell clones (P < or = 0.001), but the frequency of distant metastasis at time of diagnosis was similar. The presence of aneuploidy in at least one sample from a tumour was a significant adverse prognostic factor (P < 0.001), whereas the degree of heterogeneity had no influence on survival. The frequent heterogeneity demonstrated indicates that multiple samples must be investigated to evaluate properly the malignant character of renal cell carcinoma.     

Changes in allelic imbalances in locally advanced breast cancers after chemotherapy

In advanced breast cancers, TP53 mutation is highly predictive of complete response to high-dose epirubicin/cyclophosphamide chemotherapy. In these tumours with an altered control of genomic stability, accumulation of chemotherapy-induced genetic alterations may contribute to cell death and account for complete response. To explore the effects of chemotherapy on stability of the tumour genome, allelic profiles were obtained from microdissected tumour samples before and after chemotherapy in 29 unresponsive breast cancers (9 with TP53 mutation). Ninety-four per cent allelic profiles remained unchanged after treatment. Interestingly, 11 profiles (6%) showed important changes after treatment; allelic imbalances significantly increased (four cases) or decreased (seven cases) after chemotherapy in three distinct experiments, two of which using laser microdissected tumour cells. These genetic changes were not linked to the TP53 status, but one tumour showed complete disappearance of TP53-mutated cells in the residual tumour after treatment. Altogether, these observations carry important implications for the clonal evolution of breast cancers treated with DNA-damaging agents, as they point both to the importance of tumour heterogeneity and chemotherapy-driven selection of subclones.     

Do DNA ploidy and S-phase fraction in primary tumour predict the response to chemotherapy in metastatic breast cancer?

The relationship between the response to chemotherapy with cyclophosphamide, epirubicin and fluorouracil as well as the time to progression of metastasised breast cancer and DNA ploidy and S-phase fraction (SPF) of primary tumours was examined using paraffin-embedded tumour tissue from 81 patients. The response to chemotherapy was significantly better in patients with tumours with a high SPF, and in addition the time to progression was longer in the high-SPF group. There was no significant difference when the DNA ploidy and response to treatment were compared.     

Mutation of the p53 gene precedes aneuploid clonal divergence in colorectal carcinoma.

To establish whether p53 mutation precedes or follows clonal divergence in human colorectal carcinomas, 17 tumours were analysed at multiple sites (2-5 each) for single-strand conformation polymorphisms (SSCP) within exons 5-8 of the p53 gene. A previous study had demonstrated subclones of differing DNA ploidy in these tumours, but all showed immunocytochemical evidence for p53 stabilisation, using the monoclonal antibody PAb 1801. Mutations within exons 5-8 of p53 were identified by the presence of an abnormally migrating band in 10 of the 17 carcinomas: five in exon 5, four in exon 7 and one in exon 8. In each of these positive cases, samples from different parts of the carcinoma showed identical gel migration patterns in SSCP analysis. Similarly, the remaining seven tumours were concordant for absence of band shift across all samples of each tumour. Six SSCP-positive cases contained multiple populations differing in DNA ploidy, while four were homogeneously diploid or aneuploid throughout. Very similar proportions were observed in the SSCP-negative cases. In four positive tumours the mutation was confirmed by sequencing or through alteration of nucleotide-specific restriction enzyme cleavage. Identical mutations appeared in every sample from the same tumour. The results provide unequivocal evidence that the same mutant allele of p53 is present throughout each tumour bearing a mutation, regardless of the clonal variation identified by analysis of DNA ploidy. We conclude that in colorectal tumorigenesis mutation of p53 occurs as a single event which precedes and may facilitate the aneuploid clonal divergence of carcinomas.     

Histopathological grading and DNA ploidy as prognostic markers in metastatic prostatic cancer.

The present study compares the prognostic potential of tumour grade and DNA ploidy status in patients with advanced-stage prostatic cancer. Two outcome groups were selected on the basis of time to progression and survival after orchiectomy. A poor-outcome group consisted of 32 therapy-resistant patients who experienced disease progression during the first year after orchiectomy and subsequently death due to prostatic cancer during the following year. A good-outcome group consisted of 27 therapy-responsive patients who showed disease regression and no signs of progression during a 3 year follow-up. The primary tumours were graded twice according to WHO and Gleason classification systems by two pathologists. Final agreement between the pathologists was obtained after a consensus meeting. The analysis revealed no prognostic importance of the two histological classification systems (P = 0.62 and P = 0.70) and disclosed weak inter- and intra-observer reproducibility (kappa < 0.70). DNA ploidy analyses were performed by image cytometry on formalin-fixed, paraffin-embedded samples of the primary tumours. Overall, 48% of the tumours were diploid, 20% tetraploid and 32% anueploid. DNA ploidy status did not discriminate between the two outcome groups (P = 0.46). Histological grade and DNA ploidy showed no prognostic importance in patients with prostatic cancer and skeletal metastases.     

Flow cytometric analysis of DNA content in human ovarian cancers

A total of 155 samples from 101 patients with ovarian cancer were investigated using flow cytometry to evaluate the DNA index and the percentage of cells in the various cell cycle phases. Thirty-four samples were DNA diploid tumours, while the other 121 were DNA aneuploid tumours. The DNA index was very stable in different sites and over time in the same patient. Tumour stage and ploidy were significantly associated: stages III and IV tumour stage were more likely to be DNA aneuploid. Patients with residual tumour size at first surgery greater than 2 cm had a significantly larger number of DNA aneuploid than DNA diploid tumours. The DNA index was also related to the degree of differentiation of the tumours. The percentage of cells in the S phase of the cell cycle was significantly higher in DNA aneuploid and in poorly differentiated tumours than DNA diploid and well differentiated tumours. Multivariate analysis using the Cox model showed that the DNA index and the percentage of cells in S phase were not independent prognostic variables in this study. Prospectively collected data should be accumulated before assigning the DNA index an important role as a biological prognostic factor in ovarian cancer.     

Flow cytofluorometric evidence for the differential radioresponsiveness of aneuploid and diploid cervix tumours

We are presently involved in a prospective study of the relationship between DNA content profiles, and their changes during treatment, determined by flow cytofluorometry, and patient prognosis and response to therapy for cancer of the uterine cervix. To date, 348 patients have been included in the study over a 54-month period. Data on these patients have shown that DNA aneuploid tumours are significantly more radioresponsive than diploid cervix tumours. Analysis of the data on 213 patients with a minimum follow-up time of 15 months has, however, failed to show an overall more favourable prognosis conferred by tumour DNA aneuploidy. Analysis of the relationship between clinical stage and disease state and tumour DNA ploidy, however, suggests that aneuploid tumours metastasize to distant sites at an earlier stage of the disease than diploid tumours and local recurrence rates for diploid tumours, in late stage disease, are double those for aneuploid tumours. Improved staining procedures, and instrument modification, has also shown that cervix tumour heterogeneity is of considerably greater frequency than at first appeared to be the case (approximately 75% of DNA aneuploid tumours show heterogeneity.     

Evolving significance of prognostic markers associated with new treatment strategies in neuroblastoma

The striking differences in the natural history of clinical subgroups of neuroblastoma (NB), and the evolving therapeutic approaches for each, makes it imperative for prognostic markers to be reevaluated within individual clinical categories. At least one third of NB cases present without distant metastasis and cytotoxic therapy does not alter the natural history. We carried out a retrospective analysis of archived tumor samples. Fifty-seven of these patients had local-regional (LR) NB and were managed conservatively, initially treated with surgery alone. Among the biologic and clinical features analyzed including age, stage, histology, ploidy, MYCN, and 1p36, 1p22, 11q, 14q, 9p and 19q loss of heterozygosity (LOH) in multivariate analysis, diploidy was one of the most significant factors associated with progression-free survival and stage 4 progression. Clonal ploidy heterogeneity was common in LR NB. A predominant near-triploid clonal population was found in most cases of non-progressing LR NB tumors whereas progressing LR NB cases had a predominant diploid clone. We also reviewed the prognostic factors among 84 stage 4 NB cases treated with the N5, N6 or N7 protocols at MSKCC from 1987 to 1999. Traditional markers such as lactate dehydrogenase (LDH), ferritin, age and MYCN status were not prognostic in the univariate analysis. 11q23 and 1p22 LOH were correlated with better survival. These results highlight the evolving significance of prognostic analysis in homogeneous clinical groups undergoing similar treatments. To further characterize the gene expression profile between local-regional and metastatic NB, we carried out Microarray analysis of 41 NB tumors and 12 NB cell lines, using the Affymetrix Genechip Human Genome U95 Set. Distinct gene expression patterns between metastatic and non-metastatic NB tumors have been identified. Validation of these results and further mechanistic studies may shed new light on the biology of metastasis in human NB.     

Heterogeneity of colorectal adenocarcinomas evaluated by flow cytometry and histopathology

Flow cytometry and histopathology were utilised in evaluating 50 primary and 16 metastatic colorectal carcinomas to determine the influence of heterogeneity and proportion of dying cells on pathological assessments. A new procedure was developed for staining unfixed whole cells with acridine orange and ethidium bromide to quantify DNA and RNA content and number of dead and dying cells. Attempts were made to reduce interobserver variation in histological assessment and to determine whether flow cytometry could refine current grading and staging procedures. Interobserver variation in grading was not improved by estimating proportions of differing grades in multiple samples from individual tumours. Considerable heterogeneity was observed within tumours although this was less apparent when defining ploidy status than histological grade. No consistent differences were observed between superficial and deep parts of tumours or between primary and secondary tumours by either method of analysis. The proportion of dead and dying cells varied widely between tumours but there was no correlation with tumour grade or stage. Non-diploid tumours were not of more advanced stage or poorer histological grade than diploid tumours. Since ploidy status may be an important prognostic factor, analysis of colorectal carcinomas by flow cytometry could be of greater value than conventional grading and staging procedures.     

Co-existent anaplastic and well differentiated thyroid carcinomas: a nuclear DNA study

Clonal transformation of well differentiated follicular or papillary carcinomas has been suggested as a mechanism by which anaplastic carcinomas of the thyroid might arise. Of 126 cases of anaplastic (giant cell) carcinomas, 17 (13.5%) contained histologically well differentiated tumour foci within or adjacent to the high grade malignant anaplastic tumour. Cytophotometric DNA analysis after Feulgen staining was performed on 11 cases in order to evaluate ploidy of the anaplastic and the well differentiated tumour cells. The majority of these co-existent carcinomas (9/11) were papillary. All 11 anaplastic carcinomas demonstrated an aneuploid DNA pattern which correlated with a poor clinical outcome (7 of 11 died of disease in less than 6 months). In contrast six co-existent papillary and one co-existent follicular tumours were diploid. These data show that the co-existence of anaplastic and well differentiated carcinoma occurs only rarely and when it occurs only one third of the well differentiated tumours contain aneuploid tumour cells. This suggests that in the majority of cases of anaplastic thyroid carcinoma the malignant cells arise de novo rather than through clonal transformation of well differentiated carcinomas.     

DNA ploidy in primary testicular cancer

The DNA stemline ploidy was measured by flow cytometry (FCM) in 129 samples from paraffin-embedded primary testicular tumours (61 seminomas, 68 non-seminomas). Only one DNA stemline was found in 38 seminomas and 44 non-seminomas. Two seminomas and one non-seminoma were DNA diploid, the other tumours being non-diploid. Twenty-three seminomas and 24 non-seminomas displayed two or three DNA stemlines. The median minimal DNA index (DI) of all seminomas was significantly higher than that of all non-seminomas (1.58 vs 1.43; P: 0.008). Three seminomas removed from two monozygotic twins within 1 week had DIs of 1.66, 1.56 and 1.59. In this limited series there was no association between DNA ploidy of the primary tumour and the metastatic status for either seminomas or non-seminomas. The results support the pathogenetic model stating that at least some (if not all) non-seminomas develop from a seminoma by additional chromosomal aberration. The clinical relevance of DNA stemline ploidy has to be further evaluated in larger series.     

鼻咽癌新鲜肿瘤组织DNA倍体性与预后的关系

背景与目的:因肿瘤有生物学异质性,部分肿瘤的预后和TNM分期并不符合;寻找有效的生物学预后指标作为临床分期的补充,可为今后鼻咽癌的个体化治疗提供一个新的依据.本研究探讨初治鼻咽癌患者新鲜肿瘤组织细胞的DNA倍体性与疗效、预后的关系.方法:1999年1月至2000年2月,53例初治鼻咽癌患者进入本研究,其中单纯放疗32例,另21例患者于放疗第4周接受了一个疗程的PF方案化疗.患者治疗前均活检取新鲜肿瘤组织,用流式细胞仪进行DNA倍体检测.结果:53例患者中,二倍体32例(60.4%),异倍体21例(39.6%).不同倍体组患者的年龄、性别、临床分期、N分期、化疗与否的差异无统计学意义(P=0.695、0.657、0.088、0.972和0.335).全组患者中位随访时间73个月(12～84个月).全组5年总生存率65.61%,其中二倍体组为80.92%,异倍体组为42.86%(P=0.002);5年无远处转移生存率二倍体组为84.26%,异倍体组为44.53%(P=0.003);5年无复发生存率二倍体组为92.59%,异倍体组为72.65%(P=0.118).单因素分析结果显示,临床分期是无复发生存率的影响因素,DNA倍体性、临床分期和T分期是总生存率和无远处转移生存率的影响因素.多因素分析结果显示,与总生存率相关的独立预后因素为DNA倍体性(P=0.020)和临床分期(P=0.007),与无转移生存率相关的预后因素亦为DNA倍体性(P=0.017)和临床分期(P=0.011).结论:采用流式细胞术检测新鲜组织细胞的DNA倍体性和临床分期一样可以预测鼻咽癌患者的预后;DNA异倍体患者比二倍体患者更容易出现远处转移而导致治疗失败.     

Ploidy as a prognostic indicator in end stage squamous cell carcinoma of the head and neck region treated with cisplatinum

We measured tumour cellular DNA in 102 patients entered into two phase III trials of chemotherapy for end stage squamous carcinoma of the head and neck. The median survival of untreated patients with aneuploid tumours was 55 days compared with 224 days for patients treated with cisplatinum. This difference was highly significant. In contrast the median survival of untreated patients with diploid tumours was 74 days compared with 118 days for treated patients. Although this difference is statistically significant, the increased survival of 6 weeks is of no clinical benefit compared with the prolongation of survival of 6 months in patients with aneuploid tumours. Multivariate analysis showed that the significant predictors of survival were Karnofsky status, response to chemotherapy and ploidy.     

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.     

Distribution of non-diploid flow-cytometric DNA indices and their relation to the nodal metastasis in squamous cell carcinomas of the head and neck

Squamous cell carcinomas of the head and neck (HNSCC) evolve from diploid epithelial cells of the mucosa. At the time of diagnosis about two thirds of clinically diagnosed HNSCC are non-diploid according to flow-cytometric (FCM) analysis, indicating that during tumour progression there must be an acquisition and accumulation of chromosomal aberrations. At diagnosis one third to one half of HNSCC have clinically positive neck nodes. The objective of the present study was to see whether the progression to a metastatic phenotype is reflected in the distribution of FCM DNA ploidy in node-negative and node-positive HNSCC. The series comprised 200 patients with HNSCC. Tumour samples were obtained from diagnostic biopsies or primary surgery. A multistep preparation method and propidium iodide staining of nuclear DNA content was used for FCM. One hundred and forty one (71%) of the tumours were non-diploid. Only two tumours were hypodiploid (DNA index 0.73 and 0.93, respectively). Ten of the tumours exhibited two non-diploid stem cell lines. The frequency of non-diploidy in node-negative tumours was 65% and in node-positive ones about 80%. The frequency distribution of non-diploid DNA indices clustered in the hypotetraploid region (with a modal value of 1.71-1.74) and did not differ between node-negative and node-positive tumours. The hypothesis that the disposition to metastasis is reflected in the frequency distribution of non-diploid DNA indices could thus not be verified.     

Intratumoral heterogeneity in DNA ploidy of esophageal squamous cell carcinomas

Intratumoral heterogeneity in DNA ploidy was investigated in 23 cases of squamous cell carcinoma of the esophagus. Nuclear DNA content was determined for multiple samples taken from the same tumor, using a flow cytometric technique. The incidence of DNA aneuploidy was 87% in this series, and DNA indices ranged from 0.78 to 2.64 but most of them fell within values between 1 and 2. Of these cases ten (43.5%) showed intratumoral heterogeneity in DNA ploidy; in addition to a diploid population, one to four heterogeneous aneuploid subpopulations were discernible in the same tumor. However, morphologic variation was minimal within the same tumor. DNA index seen in metastatic lesions was identical with one of those in the primary lesion. Mechanisms responsible for intratumoral difference in DNA ploidy are also discussed from the aspect of tumor progression.     

Selection of tumour cell subpopulations occurs during cultivation of human tumours in soft agar. A DNA flow cytometric study

To examine whether selection of tumour cell subpopulations occurs during cultivation in soft agar, we compared in 23 human tumours of different histological types the DNA content of cells from colonies formed in soft agar (method of Courtenay and Mills, 1978) with that of the original tumour cells. The ploidy as well as the fraction of cells in S phase were determined from DNA histograms after staining of the nuclei with a propidium-iodide procedure and flow cytometric recordings. In 8 of 17 aneuploid tumours analysed, specific aneuploid subpopulations disappeared during cultivation or new aneuploid populations, not demonstrable in the original cell suspensions, appeared in the colonies. In 9 cases identical aneuploid populations were found in the colonies and the tumours. In one of 6 diploid tumours examined, aneuploid cell populations not revealed in the original cell suspension, were found in addition to diploid cells, whereas 5 tumours gave rise to colonies containing a purely diploid population. The results show that in a variety of human malignant tumours cultivation in soft agar may select specific aneuploid tumour cell populations.     

The influence of tumour cell DNA content on survival in colorectal cancer: a detailed analysis

We have investigated the influence of tumour cell DNA content (ploidy) on survival of 416 patients undergoing excisional surgery for colorectal cancer. Two hundred and eleven (51%) tumours had an abnormal DNA content (aneuploid or tetraploid). There was no correlation between ploidy status, sex, age and pathological stage, histological grade, tumour site, local tumour extension or assessment of curability. Patients with tumours with an abnormal DNA content had a poorer survival 68/211 (32%) than patients with near normal (diploid) DNA content 88/205 (43%) (test statistic 5.0, P = 0.02). The patient subgroups in which DNA content exerted an influence on survival were: stage B tumours (P = 0.0058), moderately differentiated tumours (P = 0.004), rectal tumours (P = 0.02), and mobile tumours (P = 0.02). Multivariant analysis showed that pathological stage, local tumour extension and DNA ploidy were all independent prognostic indicators whereas histological grade, tumour site and assessment of 'curability' were not. The influence of pathological stage, however, was much greater than that of local tumor extension or DNA ploidy. Tumour cell DNA content together with pathological stage and local tumour extension may be used in a prognostic index and may be important in planning adjuvant therapy.     

Predicting outcome for patients with node negative breast cancer: a comparative study of the value of flow cytometry and cell image analysis for determination of DNA ploidy.

This study was aimed at determining whether tumour DNA content measured by cell image analysis could provide additional prognostic information when compared to that provided by flow cytometry. Sections cut from paraffin blocks of tumours from 101 patients with node negative breast cancer were analysed by both methods and the results related to other prognostic variables and to patient relapse and overall survival. DNA ploidy measured by flow cytometry classified 46 tumours as diploid and 55 as aneuploid, whereas by cell image analysis 30 were diploid and 71 aneuploid (P less than 0.002). There were 20 tumours with discrepancies between the two methods; 18 of these were tumours with only one peak in flow analysis, but determined to be aneuploid with image analysis. DNA content as measured by both methods was significant for predicting relapse and survival by log-rank test, as were tumour histological grade, c-erbB-2 expression and tumour size. Multivariate analysis showed DNA ploidy measured by flow cytometry to be the only variable of independent significance (P less than 0.02) for both relapse and overall survival. Compared with cell image analysis, flow cytometry demonstrated a significantly higher proportion of diploid tumours, which may be related to differences in the internal standards applied to each method. We suggest that cell image analysis techniques can provide more sensitive information on the DNA content of tumour cells by direct measurement of nuclear DNA density of both normal lymphocytes and tumour cells in the same section. However, although image analysis appears to be more sensitive than flow cytometry in detecting DNA aneuploidy, the image technique appears to lack the specificity of flow cytometry in correlation with clinical outcome.     

Correlation of DNA ploidy, histopathology, stage and clinical outcome in gastric carcinoma.

The determination of nuclear DNA ploidy from paraffin-embedded specimens was performed by flow cytophotometry on 277 surgically resected primary gastric carcinomas to assess the relationship of various pathological findings and DNA content with survival. The preparation of samples was performed by a modification of Hedley's technique and the staining method of Vindelov. Eighty-nine (32%) carcinomas were DNA diploid, 69 (25%) were DNA tetraploid, and 119 (43%) were DNA aneuploid. DNA non-diploid patterns were significantly associated with macroscopic ulcerative appearance, location of the tumour in the proximal stomach, histological grade, and advanced stage of tumour. Patients with DNA non-diploid cancers, and specifically DNA aneuploid cancers, exhibited significantly poorer survival than patients with DNA diploid tumours. These data support the prognostic value of tumour DNA content in patients with resected gastric carcinoma.