P. De Angelis

Chromosomal gains and losses in primary colorectal carcinomas detected by CGH and their associations with tumour DNA ploidy, genotypes and phenotypes

SummaryComparative genomic hybridization (CGH) is used to detect amplified and/or deleted chromosomal regions in tumours by mapping their locations on normal metaphase chromosomes. Forty-five sporadic colorectal carcinomas were screened for chromosomal aberrations using direct CGH. The median number of chromosomal aberrations per tumour was 7.0 (range 0–19). Gains of 20q (67%) and losses of 18q (49%) were the most frequent aberrations. Other recurrent gains of 5p, 6p, 7, 8q, 13q, 17q, 19, X and losses of 1p, 3p, 4, 5q, 6q, 8p, 9p, 10, 15q, 17p were found in > 10% of colorectal tumours. High-level gains (ratio > 1.5) were seen only on 8q, 13q, 20 and X, and only in DNA aneuploid tumours. DNA aneuploid tumours had significantly more chromosomal aberrations (median number per tumour of 9.0) compared to diploid tumours (median of 1.0) (P < 0.0001). The median numbers of aberrations seen in DNA hyperdiploid and highly aneuploid tumours were not significantly different (8.5 and 11.0 respectively; P = 0.58). Four tumours had no detectable chromosomal aberrations and these were DNA diploid. A higher percentage of tumours from male patients showed Xq gain and 18q loss compared to tumours from female patients (P = 0.05 and 0.01 respectively). High tumour S phase fractions were associated with gain of 20q13 (P = 0.03), and low tumour apoptotic indices were associated with loss of 4q (P = 0.05). Tumours with TP53 mutations had more aberrations (median of 9.0 per tumour) compared to those without (median of 2.0) (P = 0.002), and gain of 8q23–24 and loss of 18qcen-21 were significantly associated with TP53 mutations (P = 0.04 and 0.02 respectively). Dukes’ C/D stage tumours tended to have a higher number of aberrations per tumour (median of 10.0) compared to Dukes’ B tumours (median of 3.0) (P = 0.06). The low number of aberrations observed in DNA diploid tumours compared to aneuploid tumours suggests that genomic instability and possible growth advantages in diploid tumours do not result from acquisition of gross chromosomal aberrations but rather from selection for other types of mutations. Our study is consistent with the idea that these two groups of tumours evolve along separate genetic pathways and that gross genomic instability is associated with TP53 gene aberrations.

Prognostic significance of proliferative and apoptotic markers in oral tongue squamous cell carcinomas

The prognostic impact of proliferative and apoptotic markers was studied in 85 T1-4 oral tongue squamous cell carcinomas (SCCs). Ki67 immunoreactivity and AgNOR counts, including mean AgNOR counts (mAgNOR) and the percentage of nuclei with more than one AgNOR (pAgNOR > 1), were used as proliferative parameters. The apoptotic index (AI) was assessed using the TUNEL method. Bax expression was detected immunohistochemically and scored. Bax expression correlated positively with AI (p = 0.0122). Ki67 correlated with both pAgNOR > 1 (p = 0.0042) and mAgNOR (p = 0.0189). Low Bax expression and low AI correlated significantly with the disease-free period (p = 0.0001 and p = 0.0024, respectively). High values for Ki67, pAgNOR > 1 and mAgNOR correlated with poor prognosis (p = 0.0021, p = 0.0001 and p = 0.0244, respectively). Combinations of proliferative and apoptotic parameters were stronger predictors than individual parameters (p < 0.0001). pAgNOR > 1-Bax expression appeared to be the best combination (p < 0.0001). We conclude that proliferative and apoptotic markers, especially their combinations, have prognostic value in tongue SCC.

Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype.

AIMS: Spontaneous apoptosis and expression of the apoptotic regulatory proteins Bax, Bcl-x, and Bcl-2 were investigated in 50 colorectal carcinomas. The p53 genotypes/phenotypes and BAX genotypes were also determined, and possible associations of these with apoptosis and/or with expression of the different apoptotic regulatory proteins were studied. METHODS: Terminal deoxynucleotidyl transferase (TdT) mediated dUTP labelling of DNA fragments was used to detect apoptotic tumour cells in sections and peroxidase immunohistochemistry was used to assess protein expression. p53 genotype/phenotype was determined using constant denaturant gel electrophoresis/immunoblotting and bax genotype was determined using polymerase chain reaction based methods. RESULTS: The distribution of tumour apoptotic indices was bimodal with a natural cut off at 1.0% (range, 0.0-5.4%); the median fraction of apoptotic tumour cells was 0.8%. Tumour apoptosis was not associated significantly with tumour DNA ploidy status. Normal mucosal tissue had less than 0.1% apoptotic cells. Staining intensities for Bax, Bcl-x, and Bcl-2 were strong; that is, equivalent to or greater than positive normal mucosal cells, in 11 of 50, 20 of 49, and 20 of 48 carcinomas. Frameshift mutations in the bax gene were detected in three of 42 tumours analysed, all of which were DNA diploid, and Bax protein expression in these tumours was absent or very low. Bax, Bcl-x, and Bcl-2 protein expression were not correlated with tumour apoptosis or tumour DNA ploidy status. p53 was expressed in 34 of 50 tumours and p53 gene mutations were detected in 22 of 29 p53 positive tumours analysed. Apoptosis was significantly lower in a greater number of p53 positive tumours than p53 negative tumours. In addition, Bcl-2 protein expression was significantly higher in a greater number of p53 positive tumours compared with p53 negative tumours. Bax and Bcl-x protein expression were not significantly associated with p53 phenotype/genotype. CONCLUSIONS: The results indicate that acquisition of a p53 phenotype is associated with lower spontaneous apoptosis and higher expression of Bcl-2. The results also suggest that p53 is not a major determinant for Bax expression in colorectal carcinomas in vivo.

Bax expression has prognostic significance that is enhanced when combined with AgNOR counts in glottic carcinomas

Using nucleolar organizer regions (NORs) as a proliferative marker and Bax expression as a marker for apoptosis, we have studied the individual and combined prognostic significance of these markers. Successive sections of diagnostic, formalin-fixed and paraffin-embedded specimens from 69 patients with T1-4 tumours were stained with a rabbit anti-human Bax polyclonal antibody and silver nitrate for visualization of NORs (AgNORs). After classification for staining intensity and the percentage of Bax expression, a final score resulting in four classes of increasing Bax expression was obtained. AgNOR counts were expressed as mean counts (mAgNOR) and the percentage of tumour nuclei with more than one AgNOR (pAgNOR>1). Both AgNOR parameters were grouped in three classes with increasing values. Low Bax scores correlated significantly with poor prognosis (P = 0.0106). For mAgNOR and pAgNOR>1, high values correlated with poor prognosis (P = 0.0185 and P = 0.0003 respectively). A combined parameter, for which the Bax score was subtracted from the AgNOR scores, appeared to be statistically stronger than the individual parameters (P < 0.0001). Both Bax expression and AgNOR scores, and in particular the combination of these parameters, appear to be strong prognostic markers in glottic squamous cell carcinomas.

Evaluation of ionizing radiation sensitivity markers in a panel of lymphoid cell lines.

Purpose : To examine the possible associations between radiation sensitivity to doses ≤2Gy, and such features of lymphoid cell responses as apoptosis, expression of apoptosis regulatory proteins (Bcl-2 family) and cell cycle progression in relation to biological dosimetry. Materials and methods : The cell lines examined were: Epstein-Barr virus transformed lymphoid ataxia-telangiectasia (AT) cell lines, GM00717C, homozygous, and GM00736A, heterozygous, for ATM ; human pro-B lymphoblastic leukaemia, Reh; murine L5178Y lymphoma sublines, LY-R and LY-S. Assays performed following X-irradiation with doses from 0.1 to 2 Gy were: terminal deoxyribonucleotidyl transferase (TdT) assay to measure apoptotic fraction, DNA content analysis by flow cytometry to assess cell cycle distribution, trypan blue exclusion test to determine cell viability, cytochalasin block micronucleus assay to assess cytogenetic damage, and Western blotting to detect proteins from the Bcl-2 family. Results : The cell lines in the study were of different but rather high radiation sensitivity, which was unrelated to their propensity to undergo apoptosis or micronucleus frequency. The expression of apoptotic regulatory proteins from the Bcl-2 family (constitutive and expressed 4 or 24 h after irradiation) was not related to radiation sensitivity. Conclusion : None of the simple predictive tests used in the study, alone or evaluated together was suitable for detection of radiation hypersensitivity although cells known to be hypersensitive (LY-S and GM00717C) were included in the analysis.

Retinoic acid provokes a regeneration-like proliferative response in murine epidermis

Retinoic acid (RA) is an inducer of epidermal proliferation by a mechanism of action which is not fully known. We examined the proliferative response of hairless mouse epidermis to a single topical application of different doses of RA (0.1–1000 nmol). The mitotic rate was assessed using the stathmokinetic method, and change in epidermal cell numbers were scored per microscopic vision field in tissue sections. Cell cycle parameters were measured by bivariate bromodeoxyuridine/DNA flow cytometry on isolated epidermal basal cells after pulse labelling up to 10 days after RA treatment. The results showed a dose-dependent increase in mitotic activity with a maximum at 3 days after RA application, and a dose-dependent hyperplasia with a maximum at 4 days after RA application. Cell-cycle analysis showed an immediate proliferative response after RA application similar to that following various skin irritants. Although differences in the G2 phase transit were seen, this indicates a similar mechanism of action of RA-induced epidermal proliferation and that associated with epidermal regeneration in general.

NO38 expression and nucleolar counts are correlated with cellular DNA content but not with proliferation parameters in colorectal carcinomas

AIMS: To investigate the expression of nucleolar protein NO38, to determine the numbers of nucleoli per cell, and to examine the relations of these nucleolar parameters to tumour DNA index, total cellular DNA content, S phase fraction, and Ki67 labelling index. METHODS: 36 colorectal tumours and 14 normal mucosas were studied. An anti-NO38 monoclonal antibody, 31A12, and flow cytometric analysis were used to detect expression of NO38 by means of a biotin-streptavidin-FITC (fluorescein isothiocyanate) staining method. Nucleolar counts were determined using fluorescence microscopy. Flow cytometry was used to determine tumour DNA indices and the sizes of the S phase fractions. Ki67 labelling indices were determined from tissue sections stained immunohistochemically with the MIB-1 antibody against the Ki67 nuclear protein. RESULTS: Generally, tumour cell nucleoli were larger and more irregular in shape compared with nucleoli in normal mucosal cells. DNA aneuploid and diploid tumours expressed 2.8 and 2.1 times more NO38 than normal mucosa. The mean (SD) values for nucleolar counts were higher for the DNA aneuploid tumours (3.81 (0.93)) than the diploid tumours (2.62 (0.38)) and normal mucosa (2.34 (0.37)). NO38 expression and numbers of nucleoli correlated significantly (r = 0.52, p = 0.01). There were, however, no significant correlations between these nucleolar parameters and either the sizes of tumour S phase fractions or Ki67 labelling indices. Cell cycle resolved expression of NO38 in tumours and normal mucosa demonstrated that expression increased approximately in proportion to the DNA content throughout the cell cycle. In aneuploid tumours, NO38 expression was 43% and 98% higher in S and G2 phases, respectively, compared with the G1 phase. Sorting of these populations revealed that the nucleolar count also increased as the DNA content increased but by only 29% and 47% in S and G2, respectively. Apoptotic cells lacked NO38. CONCLUSIONS: NO38 expression is higher in tumours than in normal mucosa owing to the increased DNA content and larger nucleoli in tumours; expression increases proportionally with DNA content as cells progress through the cell cycle from G1 through S and G2. However, NO38 expression does not correlate with the tumour S phase fraction or Ki67 labelling index and is lost during apoptosis. Also the results suggest that nucleoli grow in size during the cell cycle, which would account for the doubling of NO38 expression from G1 to G2, as the nucleolar count increased by only 47%.