High-resolution DNA content analysis of microbiopsy samples in oral lichen planus.

OBJECTIVES: DNA aneuploidy has been reported to be a predictor of poor prognosis in both premalignant and malignant lesions. In oral lichen planus (OLP), this hypothesis remains to be proved. This study aimed to determine the rate of occurrence of DNA aneuploidy in patients with OLP by high-resolution DNA flow cytometry. METHODS: Patients with OLP were consecutively enrolled. Tissue samples were subdivided for formalin fixation and routine histological assessment and for immediate storage at -20°C for later DNA ploidy analysis, which was performed by DAPI staining of the extracted nuclei and excitation with a UV lamp. The DNA aneuploid sublines were characterized by the DNA Index. RESULTS: A DNA aneuploid status was observed in two of 77 patients with OLP (2.6%). When considering the clinical aspect of the OLP lesions, both DNA aneuploid cases had a reticular clinical aspect. CONCLUSIONS: DNA aneuploidy is an uncommon event in OLP and less frequent compared to other non-dysplastic and non-OLP oral potentially malignant disorders. The extremely low rate of DNA aneuploidy could represent an occasional finding or reflect the low rate of malignant transformation observed in patients with OLP even if the real prognostic value of DNA ploidy analysis in patients with OLP remains to be confirmed.

Genomic aberrations in normal appearing mucosa fields distal from oral potentially malignant lesions.

OBJECTIVES: Oral fields of visually normal and non-dysplastic mucosa (ODFs) may represent the precursors of oral potentially malignant lesions (OPMLs). Aim of the study was to provide new evidence for the concept of the "field carcinogenesis" model by comparing the ODF and OPML genomic aberration profiles obtained by high resolution DNA flow cytometry (hr DNA-FCM) and array-Comparative Genomic Hybridization (a-CGH). A second aim was to investigate if specific CGH aberrations were associated with DNA aneuploidy. METHODS: Nineteen patients with single OPMLs were recruited for the study. In parallel with obtaining samples of OPML tissue from 11 leukoplakias without dysplasia (nd-OPMLs) and 8 with dysplasia (d-OPMLs), we also obtained samples from distant ODFs. DNA aneuploid nuclei detected by hr DNA-FCM were physically separated, based on DNA content, from the DNA diploid components with a DNA-FCM-Sorter. These relatively pure subpopulations of epithelial nuclei were then submitted to DNA extraction and a-CGH for a genome-wide analysis of DNA copy number aberrations (CNAs). RESULTS: The frequencies of DNA aneuploidy (DI ≠ 1) among ODFs and OPMLs were respectively 5.3% and 32%. The DI aneuploid values of ODFs and nd-OPMLs were all near-diploid (DI ≠ 1 and DI ≤ 1.4), while for d-OPMLs were high-aneuploid (DI > 1.4) in 40% of the cases. CNA averages were 1.9 in ODFs and 6.5 in OPMLs. The gain of the chromosomal region 20q13.33-qter was observed in 37% of both ODFs and corresponding OPMLs. Additional common regions included 7p22.2-pter, 11p15.5-pter and 16p13.3-pter where gains were observed. Furthermore, gains of 20q13.31-q13.33 and of 5p13.33-pter and loss of 9p21.3 were detected at high frequency (respectively, at 62.5%, 50% and 50%) only in d-OPMLs. In particular, loss at 9p21.3, gain at 5p13.33-pter and gain of 20q13.31-q13.33 were associated with DNA aneuploidy (p = 0.00004; p = 0.0005; p = 0.01). CONCLUSIONS: ODFs and OPMLs showed common CNAs in specific chromosomal regions suggesting that they may represent early events of the natural history of oral carcinogenesis according to the field effect cancerization and may contribute to the ODF-OPML transition. In addition, loss at 9p21.3 and gains at 5p13.33-pter and 20q13.31-q13.33 may contribute to DNA aneuploidization.

Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.

BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Cell kinetics analysis in patients affected by squamous cell carcinoma of the head and neck treated with primary surgery and adjuvant radiotherapy.

BACKGROUND: The increasing complexity of management strategies for patients with head and neck squamous cell carcinoma (HN-SCC) calls for the investigation of new objective prognostic parameters to subdivide patients according to the tumor's biological aggressiveness. METHODS: We evaluated in 35 HN-SCC patients the pretreatment cell kinetics parameters and DNA ploidy after in vivo infusion of bromodeoxyuridine and flow cytometric analysis. Patients were treated with radical surgery followed by conventional radiation therapy. Locoregional control data are available for follow-up times above five years. RESULTS: We found that the likelihood of locoregional control for patients with rapidly proliferating HN-SCC characterized by a short potential doubling time (Tpot <5 days) was significantly smaller than for HN-SCC patients with slow tumor proliferation (Tpot >5 days). Moreover, when patients were stratified according to DNA ploidy and Tpot value, we found that the locoregional failure rate for rapidly proliferating tumors was significantly higher for diploid HN-SCCs than for aneuploid HN-SCCs. CONCLUSION: The present data suggest that patients with resectable HN-SCC characterized by fast growth might have a worse prognosis after surgery and adjuvant conventional radiotherapy and might benefit from more aggressive radiotherapeutic modalities.

Evidence of cell kinetics as predictive factor of response to radiotherapy alone or chemoradiotherapy in patients with advanced head and neck cancer.

PURPOSE: The aim of this study was to investigate the potential clinical relevance of cell kinetics parameters to the locoregional control (LRC) and overall survival of patients affected by head and neck squamous cell carcinoma (HN-SCC) treated by conventional radiotherapy, partly accelerated radiotherapy, or alternating chemoradiotherapy. METHODS AND MATERIALS: Between January 1993 and June 1996,115 patients with HN-SCC at Stage III and IV entered the study. Multiple primary tumor biopsies were obtained 6 h after in vivo infusion of bromodeoxyuridine (BrdUrd), an analogue of thymidine that is incorporated in DNA-synthesizing cells. In vivo S-phase fraction labeling index (LI), duration of S-phase (Ts), and potential doubling time (Tpot) were obtained by analysis of the flow cytometric content of BrdUrd and DNA. Eighty-two patients were randomly assigned to receive either alternating chemoradiotherapy or partly accelerated radiotherapy, whereas 33 other matching patients received conventional radiotherapy. RESULTS: Univariate LRC analysis showed that LI value was a prognostically significant factor, independent of type of therapy. Multivariate analysis failed to show cell kinetics parameters as statistically significant factors affecting LRC probability and overall survival. However, subgroup analysis showed that LRC probability at 4 years for fast proliferating tumors characterized by a LI >/= 8% was significantly better for patients treated either with alternating chemoradiotherapy or partly accelerated radiotherapy than it was for those treated with conventional radiotherapy. Conversely, LRC probability for slow proliferating tumors (LI < 8%) treated with the three treatment modalities was similar. CONCLUSIONS: These results showed that, independent of type of treatment, pretreatment cell kinetics provided only a weak prognostic role of outcome in HN-SCC. However, this report raises the hypothesis that fast growing HN-SCC may be more likely to benefit from intensified therapy, as given in this series. Cell kinetics parameters studied by the in vivo BrdUrd/flow cytometry method might be considered predictive factors of response, providing information on which type of treatment may be selected according to tumor proliferation rate.

Ki-ras activation in vitro affects G1 and G2M cell-cycle transit times and apoptosis.

Mutant ras genes occur frequently in human neoplasia and, in particular, in pancreatic, colorectal, and lung adenocarcinomas. Recent evidence suggests that G-->T and G-->C transversions of the Ki-ras gene in codon 12 may lead to biological effects in vitro and in vivo that may be associated with an abnormal cell cycle and increased tumour aggressiveness. The role of Ki-ras activation (a G-->C transversion in codon 12, arginine for glycine) in the cell cycle and apoptosis was investigated using control and permanently transfected NIH3T3 mouse fibroblasts. Flow cytometry was used to evaluate the G1-, S- and G2M-phase transit times, the potential doubling time, the growth fraction, and the cell loss factor during asynchronous exponential growth. Apoptosis was induced in both cell lines by absence of growth factors for an extended period of time (72 h) and quantitatively evaluated using the TUNEL method coupled with flow cytometry. It was found that codon 12 G-->C Ki-ras transfected cells compared with controls, had a significant prolongation of G1 by about 50%, a reduction of the G2M transit time by 30%, and a decrease of the cell loss factor by about 90%. Apoptotic cells were about 10% in control and less than 0.5% in Ki-ras transfected cells after 72 h starvation-confluency. These data suggest that codon 12 G-->C Ki-ras activation in mouse NIH3T3 fibroblasts is associated with deregulation of checkpoint controls in the G1 and G2M phases of the cell cycle and inhibition of apoptosis. It appears plausible that these cell mechanisms are related to a proliferative advantage and that they may also be important in the progression of human tumours characterized by specific Ki-ras mutations.

Analysis of erythrocyte glycophorin-A variants by flow cytometry in lung disease patients detects the effect of tobacco smoke.

The glycophoryn A (GPA) assay evaluates somatic in vivo mutations. It is considered a cumulative biodosimeter for genotoxic exposures and is under evaluation in cancer risk assessment. GPA, a polymorphic membrane protein of the erythrocytes, determines the MN blood groups. The NO and NN variant frequencies (VF) may be detected in MN subjects (about 50% of the population) by flow cytometry using two differently labelled antibodies. We explored if GPA NO and NN VF might be relevant to the assessment of individual lung cancer risk and susceptibility, in a small population with a high prevalence of heavy tobacco smokers: 8 lung cancer patients and 16 subjects with non-malignant lung diseases associated with increased risk of lung cancer. There was a wide interindividual variability and complete overlap between non-neoplastic and neoplastic patients. A significant positive correlation was seen with smoking duration in NO VF (p = 0.04, age-adjusted). Current smokers (n = 12) displayed higher NO values than never (n = 1) or ex-smokers (n = 11), 36.3 +/- 18.2 and 21.0 +/- 13.2, respectively (p < 0.01). No association was shown with occupational exposure. The present exploratory study suggests that assessment of individual lung cancer risk and susceptibility by the GPA assay does not seem to be feasible. The assay appears to provide a biomarker of longterm exposure to tobacco smoke.

Intratumor heterogeneity of k-ras and p53 mutations among human colorectal adenomas containing early cancer.

The molecular pathways and the timing of genetic events during human colorectal carcinogenesis are still not fully understood. We have addressed the intratumor heterogeneity of the mutational status of the k-ras oncogene and of the p53 oncosuppressor gene during the adenoma-carcinoma sequence by investigating 26 human colorectal adenomas containing early cancer. An intratumor comparative analysis was obtained among the adenomatous and carcinomatous component pairs. Additionally, we have analyzed 17 adenomas having cancer in the near vicinity. The adenomatous components of the adenomas containing early cancer and the adenomas having cancer in the near vicinity had comparable frequencies for k-ras mutations (28 and 47%) but different for p53 mutations (52 and 7%, p-value = 0.01). Interestingly, the adenomatous and carcinomatous components of the adenomas containing early cancer were rarely heterogeneous for the k-ras mutational status (only in 13% of the cases) but were characterized by heterogeneity of the p53 status in 59% of the cases (p-value < 0.01). In addition, the mutations of p53 for the adenomatous components of the adenomas containing early cancer were statistically significantly associated with severe dysplasia (p-value = 0.01). Intratumor homogeneity of k-ras status during the human colorectal adenoma-carcinoma sequence suggests that the role of k-ras is more related to tumor initiation than to tumor progression. On the contrary, intratumor heterogeneity of p53 mutations indicates that the type of the p53 mutations may also be relevant for selection and expansion of new subclones leading to tumor progression.

Cell cycle variations in azoxymethane-induced rat colorectal carcinogenesis studied by flow cytometry.

Cell cycle variations and DNA aneuploidy, were investigated in different phases of azoxymethane (AOM)-induced colon carcinogenesis in rats by flow cytometry. K-ras gene mutations (transitions Gright curved arrow A) were frequently detected in aberrant crypt foci (ACF) initial pre-neoplastic lesions. The fraction of cells in the G2M-phase of the cell cycle was higher in ACF compared to the normal mucosa of control rats. A similar modification of the cell cycle was found in adenomas and adenocarcinomas but, unexpectedly, also in morphologically normal mucosa from AOM-treated animals indicating that AOM treatment permanently modifies cell cycle control in rat colon mucosa. These alterations, however, were not associated with DNA aneuploidy as reported in human sporadic colorectal cancer, suggesting that tumour development in AOM-treated rats is less dependent on aneuploidy.

The value of pretreatment cell kinetic parameters as predictors for radiotherapy outcome in head and neck cancer: a multicenter analysis.

PURPOSE: The aim of this study was to assess the potential of pre-treatment cell kinetic parameters to predict outcome in head and neck cancer patients treated by conventional radiotherapy. MATERIALS AND METHODS: Data from 11 different centers were pooled. Inclusion criteria were such that the patients received radiotherapy alone, and that the radiotherapy was given in an overall time of at least 6 weeks with a dose of at least 60 Gy. All patients received a tracer dose of either iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) intravenously prior to treatment and a tumor biopsy was taken several hours later. The cell kinetic parameters labeling index (LI), DNA synthesis time (Ts) and potential doubling time (Tpot) were subsequently calculated from flow cytometry data, obtained on the biopsies using antibodies against I/BrdUrd incorporated into DNA. Each center carried out their own flow cytometry analysis. RESULTS: From the 11 centers, a total of 476 patients conforming to the inclusion criteria were analyzed. Median values for overall time and total dose were 49 days and 69 Gy, respectively. Fifty one percent of patients had local recurrences and 53% patients had died, the majority from their disease. Median follow-up was 20 months; being 30 months for surviving patients. Multivariate analysis revealed that T-stage, maximum tumor diameter, differentiation grade, N-stage, tumor localization and overall time correlated with locoregional control, in decreasing order of significance. For the cell kinetic parameters, univariate analysis showed that only LI was significantly associated with local control (P=0.02), with higher values correlating with a worse outcome. Ts showed some evidence that patients with longer values did worse, but this was not significant (P=0.06). Tpot showed no trend (P=0.8). When assessing survival in a univariate analysis, neither LI nor Tpot associated with outcome (P=0.4, 0.4, respectively). Surprisingly, Ts did correlate with survival, with longer values being worse (P=0.02). In the multivariate analysis of local control, LI lost its significance (P=0.16). CONCLUSIONS: The only pretreatment kinetic parameter for which some evidence was found for an association with local control (the best end-point for testing the present hypothesis) was LI, not Tpot, and this evidence disappeared in a multivariate analysis. It therefore appears that pretreatment cell kinetic measurements carried out using flow cytometry, only provide a relatively weak predictor of outcome after radiotherapy in head and neck cancer.

Intratumor heterogeneity of chromosome 1, 7, 17, and 18 aneusomies obtained by FISH and association with flow cytometric DNA index in human colorectal adenocarcinomas.

BACKGROUND: The origin and evolution of somatic chromosome aberrations in colorectal cancer is still poorly understood. The data in the literature suggest that some specific chromosome aberrations are more common. It is not known, however, if there is a correlation of these with near-diploid and high aneuploidy previously proposed to be a characteristic of the adenoma-carcinoma sequence. METHODS: Chromosome 1, 7, 17 and 18 numerical aberrations and 1p deletions were evaluated by fluorescence in situ hybridization analysis for 20 human sporadic colorectal adenocarcinomas in 70 distinct tumor sectors and correlated with flow cytometric DNA index (DI) values. RESULTS: Aneusomy for at least one of the investigated chromosomes was observed in 60 of 70 tumor sectors corresponding to 19 of 20 adenocarcinomas (95%). Deletions at 1p, observed in 8 of 18 adenocarcinomas (44%), were intratumor homogeneous in 7 of 8 tumors. In contrast, the other aberrations were intratumor heterogeneous. Aneusomies of chromosomes 1, 7, and 17 were strongly associated with DNA high aneuploidy (DI > or = 1.4), whereas aneusomy of chromosome 18 and 1p deletions were equally common among DNA diploid and near-diploid tumors (DI < 1.4 and DI not equal to 1). CONCLUSIONS: Overall, these data suggest the existence of different aneuploidization routes correlated with specific chromosome aberrations. In addition, intratumor homogeneity of 1p deletions appears to be an indication of early occurrence or strong selection. We also suggest that tumors with monosomies and in particular monosomies-trisomies for the same chromosomes support a model of aneuploidization and chromosome instability during the colorectal tumor progression based on loss of symmetry during chromosome segregation (Giaretti: Lab Invest 71:904-910, 1994).

K-ras2 activation and genome instability increase proliferation and size of FAP adenomas.

The possible role of K-ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP) adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%), low grade dysplasia (63%), and were preferentially located in the right colon (60%). Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K-ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO) hybridization, while flow cytometry (FCM) was used for evaluating degree of DNA ploidy and S-phase fraction. Overall, incidences of K-ras2 mutations, DNA aneuploidy and high S-phase values (>7.2%) were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K-ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K-ras2 and size, DNA ploidy and size and K-ras2 and S-phase (p < 0.001). In particular, among the wild type K-ras2 adenomas, high S-phase values were detected in 8% of the cases versus 57% among the K-ras2 mutated adenomas (p = 0.0005). The present series of FAP adenomas indicates that K-ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.

p53 mutations and DNA ploidy in colorectal adenocarcinomas.

The p53 tumour suppressor gene has an important role in the the maintenance of genome stability and its mutational inactivation may be at the origin of aneuploidy in cancer cells. The aim of this study was to determine whether p53 mutations were associated to DNA aneuploidy, as assessed by flow cytometry, in colorectal adenocarcinomas. Analysis of p53 mutations spectrum of the sorted nuclei was done by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. Overall, we studied 20 adenocarcinomas, the corresponding control mucosa, and 7 lymph node metastases. Five tumours (25%) were DNA diploid, while 15 tumours (75%) were composed of DNA aneuploid and diploid subpopulations. DNA diploid control mucosa and adenocarcinomas showed no p53 mutations, while 60% of the tumours with DNA aneuploidy had p53 mutations. Therefore, p53 mutations occurred significantly more often in DNA aneuploid than in DNA diploid tumours (p < 0.04, Fisher's exact test). Incidences of DNA aneuploidy and p53 mutations in lymph node metastases were 60 and 86%, respectively. In all tumours showing a p53 mutation, the wild-type allele was not or only bearly visible in DNA aneuploid cells suggesting that, in such cells, aneuploidy is accompanied by complete p53 functional inactivation. The present observations suggest that p53 mutations may have a role in the origin of aneuploidy at late stages of colorectal carcinogenesis.

Specific K-ras2 mutations in human sporadic colorectal adenomas are associated with DNA near-diploid aneuploidy and inhibition of proliferation.

Recent studies indicate that p21ras proteins mediate their multiple cell functions through interactions with multiple effectors and that the number of new effectors is growing. We recently reported that K-ras2 mutations in human colorectal adenomas were associated with chromosome instability and proliferation changes. In the present study, we extend these previous observations. Hereditary and multiple (n > or = 5) adenomas and adenomas with early cancer were excluded. Dysplasia was moderate in 91 cases and high in 25, and the median adenoma size was 1.5 cm. K-ras2 spectrum analysis was done by sequence-specific oligonucleotide hybridization using nuclear suspensions provided by analysis and sorting of multiparameter flow cytometry. In particular, tissue inflammatory cells were separated for DNA diploid tumors, whereas DNA aneuploid epithelial subclones were analyzed separately. K-ras2 mutations and DNA aneuploidy were both detected in 29 of 116 (25%) cases. DNA aneuploid index was in the near-diploid region in the majority of cases. DNA aneuploidy was strongly associated with G-->C/T transversions. An association was also found between low S-phase values and G-->A transitions. These findings were confirmed using multivariate logistic regression analysis to account for the effects of size, dysplasia, site, type, age, and sex. These data suggest that specific K-ras2 mutations in a subgroup of human sporadic colorectal adenomas play a role in chromosome instability and, contrary to expectations, are associated with inhibition of proliferation.

Intratumor distribution of 1p deletions in human colorectal adenocarcinoma is commonly homogeneous: indirect evidence of early involvement in colorectal tumorigenesis.

BACKGROUND: Cytogenetics and molecular biology studies have indicated that a large subset of human colorectal adenocarcinomas have distal 1p chromosome arm deletions. The aim of this study was to evaluate the intratumor distribution of 1p deletions under the assumption that homogeneity is an indication of early occurrence. METHODS: Seventy-nine histologically selected primary sectors (40 superficial and 39 deep) and 3 lymph node metastases obtained from 20 human sporadic adenocarcinomas were analyzed. Interphase two-color fluorescence in situ hybridization (FISH) was applied to cytocentrifuged nuclei using a centromeric probe for chromosome 1 and a telomeric probe mapping to the 1p36 band. RESULTS: Deletions at 1p were observed in 35 of 82 tumor samples corresponding to 9 of 20 adenocarcinomas analyzed (45%). Seven of the 9 adenocarcinomas with 1p deletions showed an intratumor presence of these aberrations in all the different tumor sectors. CONCLUSIONS: These data, acquired by FISH interphase cytogenetics, confirm that 1p deletions in colorectal adenocarcinoma are common and suggest that this structural chromosomal aberration occurs mainly as an early event in colorectal tumorigenesis.

Correlation between 1p deletions and aneusomy in human colorectal adenomas.

Human sporadic colorectal adenomas are characterized by a relatively high occurrence of aneuploidy. Similarly, 1p deletions have been reported to be an early event in colorectal tumorigenesis, while chromosome 7, 17 and 18 gain/losses were also found. The present study investigated 1p deletions, the numerical aberrations of chromosomes 1, 7, 17 and 18, and the nuclear DNA content as obtained by flow cytometry in a series of 34 human sporadic colorectal adenomas. From these adenomas, 51 intra-adenoma regions were microdissected according to 2 degrees of dysplasia and presence of foci of early cancer. Isolated epithelial nuclei were analyzed by fluorescence in situ hybridization interphase cytogenetics using centromeric probes for chromosomes 7, 17 and 18 and, in a double-target analysis, a centromeric probe for chromosome 1 simultaneously with a telomeric probe mapping to the 1p36 band. Aneuploidy incidence due to presence of numerical aberrations for at least one among the investigated chromosomes and/or abnormal flow-cytometric DNA content was 35%, while 1p deletion incidence was 38%. The correlation of 1p deletions with aneuploidy was statistically highly significant (p = 0.003), suggesting that loss of genes in this region may be implicated in chromosome instability.

Preliminary characterization of a monoclonal antibody (AS-2) against cell cycle related proteins.

A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described. AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.

Consensus report of the task force on standardisation of DNA flow cytometry in clinical pathology. DNA Flow Cytometry Task Force of the European Society for Analytical Cellular Pathology.

Guidelines are given to assist the standardisation of DNA flow cytometry in clinical pathology. They have been agreed by a group of twelve scientists from nine European countries.