Fré T. Bosman

Proliferation and apoptosis in proliferative lesions of the colon and rectum

Abstract Classically, neoplasia has been considered to be primarily a disturbance in the regulation of proliferation, but it is now clear that programmed cell death is dysregulated as well as proliferation. The genes that are implicated in the regulation of these processes, such as p53, c-myc and bcl-2, are often also altered in neoplasms. We have studied proliferation and programmed cell death in hyperplastic polyps, adenomas, carcinomas in adenomas and adenocarcinomas of the colorectum, using the MIB-1 antibody which recognizes the Ki-67 proliferation related antigen, and an in situ nick-end labelling procedure for histochemical labelling of proliferating and apoptotic cells. In addition, immunohistochemistry was used to study the expression of the p53, c-myc and bcl-2 proteins. The material studied consisted of 12 samples of normal mucosa, 8 hyperplastic polyps, 39 adenomas with different degrees of dysplasia and including 3 that carried a carcinoma, and 10 adenocarcinomas, all formalin fixed and paraffin embedded. The Ki-67 index indicated that proliferation increased progressively in hyperplasia, through different degrees of dysplasia in adenoma, to reach the highest level (Ki-67 index of 50%) in adenocarcinoma. Apoptosis also increased in hyperplastic polyps and in adenomas, but decreased significantly in adenocarcinomas. p53 Labelling was seen in 77% of the carcinomas but in only 3% of the adenomas. Expression of c-myc increased in adenomas and carcinomas. Furthermore, a shift from predominantly nuclear to predominantly cytoplasmic expression was seen in progressive neoplasms. Expression of bcl-2 was increased in an occasional hyperplastic polyp, but was increased markedly in almost all adenomas. Strikingly, in the adenomas with a carcinoma, the carcinoma showed weaker bcl-2 expression than the adenoma. In 20% of the carcinomas some bcl-2 staining was seen but this was less extensive than in the adenomas. Our findings indicate that in the progression from adenoma to carcinoma both increased proliferation and decreased apoptosis occur. This is paralleled by an increased expression of p53 and an increased and predominantly cytoplasmic expression of c-myc, but a decreased expression of bcl-2. This decreased bcl-2 expression does not lead to an increase in apoptotic activity.

Prognostic value of RET proto-oncogene point mutations in malignant and benign, sporadic phaeochromocytomas

Somatic mutations in the RET proto‐oncogene are involved in the pathogenesis of an important subset (40–60%) of sporadic medullary thyroid carcinomas (MTCs) and less frequently (0–31%) in benign, sporadic phaeochromocytomas. Since limited data exist regarding the significance of somatic RET mutations in malignant phaeochromocytomas, we analysed a multicentre series of proven malignant (i.e., metastasised) phaeochromocytomas. Analogous with MTCs, where RET mutations lead to an aggressive behaviour, we hypothesised that somatic mutations would occur more frequently in malignant than in benign phaeochromocytomas. Paraffin‐embedded tissue was available from 29 malignant and 27 benign phaeochromocytomas. Exons 10, 11 and 16 were analysed by non‐radioactive single‐strand conformation polymorphism, heteroduplex gel electrophoresis, restriction enzyme digestion and aberrant band patterns by non‐isotopic sequencing. In only 1 of 29 malignant phaeochromocytomas was a mis‐sense mutation found (at codon 634 of exon 11), whereas in 15% (4/27) of the benign tumours a point mutation was detected (in 3 tumours in exon 16 at codon 918 and in 1 tumour in exon 10 at codon 618). Absence of these mutations in non‐tumourous DNA proved their somatic origin. Contrary to what has been reported for MTCs, oncogenic RET mutations are not associated with an aggressive tumour behaviour in sporadic phaeochromocytomas. Int. J. Cancer (Pred. Oncol.) 79:537–540, 1998.© 1998 Wiley‐Liss, Inc.

Immunohistochemical, morphological and ultrastructural resemblance between dendritic cells and folliculo-stellate cells in normal human and rat anterior pituitaries

Immunolabeling of cryo‐sections of human anterior pituitaries obtained at autopsy, and of cryo‐sections of freshly prepared rat anterior pituitaries, with a panel of monoclonal antibodies against markers of the monocyte/dendritic cell/macrophage lineage, reveals in both species a characteristic pattern of immunopositive cells, among which many cells with dendritic phenotype are found. Cells characterized by marker expression of MHC‐class II determinants and a dendritic morphology are present in both human and rat anterior pituitary. Markers characteristic of dendritic cells such as the L25 antigen and the OX6 antigen were present in anterior pituitaries from human and rat respectively. The population of MHC‐class II expressing dendritic cells of the rat anterior pituitary is compared at the ultrastructural level with the folliculo‐stellate cell population, which cell type has been previously characterized by its distinctive ultrastructure and immunopositivity for the S100 protein. Using immuno‐electron microscopy of rat anterior pituitaries fixed with periodate‐lysine‐paraformaldehyde, we were able to distinguish non‐granulated cells expressing MHC‐class II determinants, whereas no MHC‐class II expression was found in the granulated endocrine cells. Using double immunolabeling of cryo‐sections of these rat AP with 25 nm and 15 nm gold labels, we demonstrated an overlap between the populations of MHC‐class II‐expressing and S100 protein‐expressing cells. Furthermore, MHC‐class II‐expressing and S100‐positive cells showed ultrastructural characteristics that have been previously ascribed to folliculo‐stellate cells. At the light microscopical level in the rat AP, a proportion of 10 to 20% of the S100‐positive cells was found immunopositive for the MHC‐class II marker OX6. In the human AP, S100‐positive folliculo‐stellate cells and cells expressing the leukocyte common antigen CD45 were found to occupy predominantly different tissue compartments in the human anterior pituitary, namely the epithelial parenchyme cords and perivascular compartments respectively. A proportion of CD45+ cells was found in the parenchyme compartment and, vice versa, indicating an overlap of the tissue compartments in which both cell types occur. However, at the light microscopical level we could not find cells expressing both the S100 and CD45 marker. The present finding of a proportion of S100‐positive pituitary cells with ultrastructural and immunohistochemical characteristics of both dendritic cells and folliculo‐stellate cells, confirms the suggested heterogeneity of the latter cell group with respect to their ultrastructural phenotype and putative function. The possibility of a myeloid origin of part of the folliculo‐stellate cell group in the AP, is discussed and might elucidate some of the discrepancies in the literature concerning the embryological origin of this cell group.

Southern and dot blot analysis of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas

SummaryTwo extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffinembedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA.Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.

Interphase in situ hybridization to disaggregated and intact tissue specimens of prostatic adenocarcinomas

A comparative study was performed of interphase in situ hybridization (ISH) to deparaffinized 4-μm tissue sections and nuclear suspensions from eight prostatic adenocarcinomas, as well as one normal prostatic control. Whole nuclear suspensions were derived from the same tumor areas to evaluate differences of ISH to truncated versus whole nuclei. DNA probes specific for the centromeres of chromosome 1, 7, 8, 10, and Y were used for detection of numerical chromosomal changes and aneuploidy. In six adenocarcinomas chromosome aberrations (+7, +8, −8, −10, −Y) were seen. However, ISH to sections revealed focal aberrations (−10, −Y) in four cases that could not be distinguished in the suspensions. Chromosomal alterations occurring in larger tumor areas were also detected in the nuclear suspensions. Chromosome copy number changes, especially gains, were better discriminated in the nuclear suspensions. The rate of ISH aneuploidy seen in nuclear suspensions corresponded with that observed in the tissue sections (P<0.01). Ploidy patterns as assessed by ISH to sections and nuclear suspensions were in concordance with DNA flow cytometry (bothP<0.001). We conclude that both section and suspension ISH were able to accurately detect aneuploidy and numerical chromosomal aberrations occurring in larger histological areas. However, section ISH was also capable of revealing (small) focal cytogenetic abnormalities, due to a precise analysis of only target cells. Focal abnormalities were not detected by suspension ISH, probably due to an admixture of non-aberrant tumor cells and stromal elements.

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion

SummaryA new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoys, Bouins or Zambonis fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37° C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.

Longitudinal evaluation of cytogenetic aberrations in prostatic cancer : Tumours that recur in time display an intermediate genetic status between non-persistent and metastatic tumours

Only limited data are available on chromosomes specifically involved in prostatic tumour progression. This study has evaluated the cytogenetic status of primary prostatic carcinomas, local tumour recurrences, and distant metastases, representing different time points in prostatic tumour progression. Interphase in situ hybridization (ISH) was applied with a set of (peri) centromeric DNA probes, specific for chromosomes 1, 7, 8 and Y, to routinely processed tissue sections of 73 tumour specimens from 32 patients. Longitudinal evaluation was possible in 11 cases with local recurrence and nine cases with distant metastases. The remaining 12 patients showed no evidence of local recurrence or distant metastasis after radical prostatectomy on follow‐up (mean 60·5 months) and served as a reference. Numerical aberrations of at least one chromosome were found in 27 per cent of the local recurrences and 56 per cent of the distant metastases. In decreasing order of frequency, +8, +7, and −Y were observed in the recurrences, and +8, +7, −Y, and +1 in the distant metastases. Evaluation of the corresponding primary tumour tissue of the recurrence group showed numerical aberrations in 45 per cent of cases. The aberrations found were, in decreasing order of frequency, −Y, +7, and +8. In the concomitant primary tumour tissue of the distant metastasis group, numerical aberrations were detected in 67 per cent of cases. The aberrations most frequently encountered were +8, −Y, followed by +7. In four cases, a concordance was found between the primary tumour and its recurrence or distant metastasis. Discrepancies might have been caused by cytogenetic heterogeneity. Comparison of the primary tumour tissue of the reference, the recurrence, and the distant metastasis groups showed a significant increase for the percentage of cases with numerical aberrations (Ptrend=0·02). Likewise, a trend was seen for gain of chromosome 7 and/or 8 (Ptrend<0·05). The number of DNA aneuploid tumours also increased in these different groups (Ptrend=0·03). These data suggest that cancers which recur in time display an intermediate position between tumours of disease‐free patients and metastatic cancers.

Cell Surface Adenosine Deaminase (ADA) and its Complexing Protein (ADCP) in Human T-Lymphoid Cells

Adenosine deaminase (ADA, EC no. 3.5.4.4), the enzyme which converts adenosine and deoxyadenosine to inosine and deoxyinosine respectively, plays an important role in the development of the immune system. The enzyme is probably involved in T-lymphocyte differentiation since a deficiency of the enzyme is associated with severe combined immunodeficiency disease.1

Recurrence and survival after resection of adenocarcinoma of the gastric cardia

In a retrospective study, the results after resection of carcinoma of the gastric cardia in the era without neoadjuvant therapy or extended lymph node dissection were evaluated. All 184 patients who underwent resection between January 1983 and December 1993 were included. Recurrence of disease, survival and prognostic factors were determined. The overall cumulative 5-year recurrence rate was 71% and the survival rate 23%. Multivariate analysis identified locoregional lymph node and distant metastases as the crucial prognosticators of recurrence of disease and survival. These results were similar to those from a previous study concerning our patients operated during the years 1983-88. The prognosis of a resected cardiacarcinoma has remained unchanged in our hands over the past 10 years. These results stress the importance of exploring new ways, such as the use of new diagnostic tools, to optimize preoperative patient selection and more aggressive treatment regimens to improve final outcome.

Comparison of automated and manual analysis of interphase in situ hybridization signals in tissue sections and nuclear suspensions

In this study we compared visual and automated analyses of interphase in situ hybridization (ISH) signals in five prostatic tumor specimens and one normal prostate sample, both in tissue sections and nuclear suspensions. The advantage of tissue sections is preservation of tissue morphology allowing precise analysis of tumor cells only. The advantage of nuclear suspensions is easier access to automated analysis, due to their disaggregated and dispersed cellular appearance. The samples were hybridized with probes for the (peri)centromeric regions of chromosome 1 and Y. The number of ISH signals per nucleus was counted both manually and automatically by means of a commercially available image analysis system. After image analysis the results were interactively corrected using a gallery display. The automatic and manual counts, before and after interactive correction, were then statistically evaluated. We found no significant differences in overall distributions between the automated and the manual counts, before as well as after correction. This was observed for both tissue sections and cellular suspensions. It is therefore concluded that automated analysis of ISH signals is feasible in both nuclear suspensions and in tissue sections, despite a low percentage of nuclei that could be measured on the latter.