Radiosensitivity of mesothelioma cell lines.

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters alpha and beta of the linear quadratic model (LQ-model) and mean inactivation dose (D(MID)) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean alpha value was 0.26 (range 0.48 - 0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The alpha/beta ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h.

Mouse subrenal capsule assay chemosensitivity and DNA flow cytometry parameters of human melanoma metastases.

Chemosensitivity was analyzed by mouse subrenal capsule assay (SRCA) in 103 human melanoma metastases 79 of which were also analyzed by DNA flow cytometry. The most effective combination was dacarbazine, vincristine, and carmustine (DOB), followed by cisplatin plus etoposide (Plat-VP16). By the original criteria of the assay, 35% of tumors were sensitive to DOB treatment while 15% responded to Plat-VP16. Chemosensitivity of DNA diploid and aneuploid tumors showed no significant difference; 35% of the aneuploid tumors were sensitive to DOB and 19% to Plat-VP16, whereas 41% and 13% respectively of the diploid tumors were sensitive. An association between DNA index and chemosensitivity was observed. Growth of the implant was observed in 44% of tumors with a DNA index above 1.5 receiving DOB treatment, whereas only 12% of tumors with an aneuploid DNA index < or = 1.5 grew. No significant difference was observed in the SPF of chemotherapy sensitive and insensitive tumors, though a tendency to lower SPF was observed in sensitive tumors.

Tumour growth rate and DNA flow cytometry parameters as prognostic factors in metastatic melanoma.

The prognostic value of flow cytometric parameters and tumour growth rate of melanoma metastases under the mouse renal capsule was investigated for tumours from 117 consecutive patients referred to the Helsinki University Central Hospital Melanoma Team. DNA flow cytometry (FCM) was interpretable for the tumours of 114 patients, and growth rate analysis for 82 patients, both results being available from 79 patients. Thirty-six percent of the tumours were DNA diploid and 64% DNA aneuploid. Tumour ploidy and S-phase fraction were shown by multivariate Cox model analysis to be independent prognostic variables and major determinants of survival after first recurrence. Patients with DNA diploid or aneuploid tumours survived a median 16 and 27 months, respectively. A high growth rate of tumour sample in vivo under the mouse renal capsule tended to be a sign of poor prognosis, although not reaching statistical significance. Combining the results of FCM, tumour growth rate and TNM stage, we propose a highly efficient prognostic scoring method. Patients with a score above 0.75 had a median survival of 11 months compared to 30 months among patients scoring under 0.75 (P less than 0.0001). This score was the most significant (P less than 0.0001) prognostic factor in the Cox model when TNM stage, age, ploidy, SPF, and tumour growth rate were analysed as covariates.

Interphase cytogenetics on paraffin sections of malignant pleural mesothelioma. A comparison to conventional karyotyping and flow cytometric studies.

We performed in situ hybridization (ISH) studies of malignant pleural mesotheliomas to detect numerical aberrations of chromosomes 1 and 7 in interphase nuclei of paraffin sections of 13 cases that had been analyzed previously by conventional karyotyping and flow cytometry. The hybridizations were performed with the biotin-labeled probes recognizing repetitive DNA sequences in the (peri)centromeric regions of chromosomes 1 (1q12) and 7(7cen). Application of histologic sections allowed us to analyze the tumor cells only. Comparison of the karyotype and ISH studies showed that the same chromosome copy numbers were detectable by both methods in 13 (chromosome 1) and in 12 (chromosome 7) cases evaluable by ISH. DNA indexes determined in the paraffin-embedded tumor material corresponded with the ISH findings. As compared with karyotype analysis, ISH showed a larger heterogeneity in chromosome copy numbers. The results can be divided into three groups: 1) Monosomy or disomy of chromosomes 1 and 7 was detected by both methods in two cases; 2) in four cases, disomy of both chromosome 1 and 7 was observed in most of the cells by ISH analysis, and karyotype analysis had shown clear polyploidization in three of these cases; 3) in seven cases, supernumerary copies of chromosomes 1 and/or 7 were present in an evident fraction (27-80%) of the cells analyzed by ISH, and karyotype analysis confirmed the aberrant copy numbers in five of these cases. On the other hand, ISH showed copy numbers not detected by karyotype analysis in six of the seven cases. Thus, by combining karyotype and interphase cytogenetic studies, complementary information about chromosomal aberrations in mesothelioma is obtained.

Comparison of DNA and karyotype ploidy in malignant mesothelioma.

The correlation between flow cytometric analysis and classical karyotyping was examined in malignant mesothelioma. Flow cytometry was used to determine the DNA ploidy mainly from paraffin blocks of 31 mesotheliomas, of which the S-phase fraction (SPF) could be defined in 17 tumors. Chromosomal results were available from 27 of these tumors. For the comparisons, both the modal (the most common) and the mean (the average) chromosome numbers were determined. A significant correlation was observed between ploidy pattern, i.e., DNA indexes and modal as well as mean chromosome numbers (p = 0.004 and p = 0.006, respectively). Otherwise the comparison between the mean chromosome numbers and DNA index proved more relevant. Although the majority of the tumors (16 of 27) had a normal modal chromosome number of 46, only 5 tumors had a normal mean chromosome number, and the remaining 22 had an abnormal number, reflecting the divergent chromosomal abnormalities. Furthermore, comparison of SPF with mean chromosome numbers disclosed a parabolic relationship; i.e., when mean chromosome number corresponded to diploid, near-diploid, or near-tetraploid cell, SPF was low, but SPF was at the maximum when mean chromosome number ranged between 60 and 63. Because SPF is an indicator of cell proliferation, this might suggest that the number of chromosomes as such also corresponds with the growth characteristics of malignant mesothelioma.

DNA aneuploidy and low S-phase fraction as favourable prognostic signs in metastatic melanoma.

The prognostic value of cellular DNA content in melanoma metastases was investigated by flow cytometric analysis of fresh or paraffin-embedded tumour blocks from 95 consecutive patients referred to the Helsinki University Central Hospital Melanoma Team. Thirty-three per cent of the tumours were DNA diploid and 67% DNA aneuploid. S-phase fractions were lower in DNA diploid than in DNA aneuploid tumours (10.7% and 17.6%). Tumour ploidy and S-phase fraction were shown by multivariate Cox model analysis to be independent prognostic variables and major determinants of survival after first recurrence. Surprisingly, patients with DNA aneuploid tumours and with tumours with low SPF survived significantly longer than those with DNA diploid or high SPF tumours. This exceptional finding of favourable prognosis for DNA aneuploid tumours was more prominent among patients receiving intensive systemic therapy and among patients with stage IV disease, probably indicating a tendency for DNA aneuploid tumours to have higher sensitivity to systemic therapy.

Serum carcinoembryonic antigen and DNA ploidy in colorectal carcinoma. A prospective study.

We have analysed the relationship between carcinoembryonic antigen (CEA) and DNA ploidy prospectively in 130 colorectal carcinoma patients. CEA was elevated preoperatively significantly more often in patients with DNA-aneuploid tumours than in DNA-diploid or DNA-tetraploid tumours--that is, in 48% (36 of 75) of patients with aneuploid tumours, in 34% (14 of 41) of patients with diploid tumours, but only in 14% (2 of 14) of patients with tetraploid tumours (p less than 0.05). Aneuploid tumours had an elevated CEA level in 38% of stage A-B disease and in 61% of stage C-D disease. The elevated CEA values (greater than or equal to 5.0 micrograms/l) correlated with tumour stage in patients with aneuploid tumours but not in patients with diploid tumours. Whereas CEA is a suitable marker for aneuploid carcinomas, other more sensitive tumour markers should be sought for diploid and also for tetraploid tumours. If such markers are found, flow cytometry could provide the most important information in selecting individual follow-up programmes for colorectal cancer patients.

The effect of fractionated irradiation on cell kinetics.

The effects of single and split-dose irradiation were compared by in vitro experiments on HeLa cells. Changes in rate of cell proliferation were detected by flow cytometry, simultaneously determining the DNA content and the bromodeoxyuridine incorporation of individual cells. Cell cultures were irradiated with either a single dose of 1-6 Gy or with a corresponding dose divided into multiple fractions given at 1-6-h intervals. A dose-dependent accumulation of cells in G2/M phase was observed. The method was sensitive enough for the detection of G2/M block even after 1 Gy. The block disappeared completely within a 24-h follow-up time at dose levels up to 3 Gy. Interestingly, no differences in cell kinetics were observed between the single and split-dose regiments. This approach proves to be valuable in evaluating novel fractionation models and the effects of radiation on the cell kinetics of human tumor cells.

DNA ploidy pattern and S-phase characteristics of metastatic melanoma.

Samples of 130 metastatic melanomas from 92 patients were analyzed by DNA flow cytometry. DNA aneuploidy was observed in 67% of the patients. DNA indices were evenly distributed from 0.6 to 2.6 Tumors originating from primary lesions in the lower extremities were more frequently DNA aneuploid than those of other sites. S-phase fraction (SPF) was evaluable from 73 tumors. DNA aneuploid tumors had a significantly higher SPF than diploid tumors, and females had a higher SPF than males. Furthermore, distant metastases had a higher SPF than metastases in regional lymph nodes and in transit metastases, probably indicating a higher growth potential in metastases spreading to distant sites.

Diploid predominance and prognostic significance of S-phase cells in malignant mesothelioma.

70 histologically verified, malignant mesotheliomas were analysed by flow cytometry for DNA content and S-phase fraction (SPF) of tumour cells. 60% (42/70) were DNA diploid. 18 of the 28 aneuploid tumours were near-diploid with DNA indices of 1.3 or less. SPF could be calculated in 51 cases. SPF was significantly higher in aneuploid (median 16.0%) than in diploid tumours (median 5.6%). DNA ploidy was not a prognostic determinant; survival was the same for both aneuploid and diploid tumours. SPF, however, was significantly correlated (P = 0.039) with prognosis. Patients who had tumours with a low SPF survived almost twice as long as those with a high SPF. Thus malignant mesothelioma has a peculiar DNA ploidy pattern compared with many other solid tumours, with a predominance of diploid or near-diploid type cells. As in many other tumours, SPF may be used as a clinically relevant prognostic indicator.

The prognostic value of DNA-ploidy in colorectal carcinoma: a prospective study.

One hundred and fifty-seven patients with usual colorectal cancer were analysed prospectively for DNA-ploidy, DNA-index and S-phase fraction (SPF) using flow cytometry. An abnormal DNA-stemline was observed in 68% of tumours. The patients have been followed for a median of 36 months. In univariate analysis, tumour stage was the most significant prognostic factor. After excluding patients with stage D disease, DNA-aneuploidy was significantly associated with a shorter survival and a shorter disease free survival. SPF, however, did not correlate with prognosis. In multiple samples from the same tumour there was on average a 29% difference between the highest and the lowest SPF indicating considerable heterogeneity in proliferative activity within the tumours. In diploid tumours the variation was even higher. Patients with proximal tumours as well as female patients had DNA-diploid tumours more often than the others. This may indicate that there are different, so far unknown, aetiological factors leading to different types of ploidy pattern.

Comparison of DNA and karyotype aneuploidy in malignant lymphomas.

Tumors from 48 patients with non-Hodgkin's lymphoma were examined for flow cytometric DNA ploidy and chromosome constitution to determine the degree of concordance of these two methods. Histologically, there were 24 low-grade, 19 intermediate, and 5 high-grade lymphomas. Flow cytometry revealed an aneuploid cell population in 19% of the cases. The mean DNA index of the aneuploid tumors was 1.58 +/- 0.71. The frequency of DNA aneuploidy was only slightly higher (23%) in intermediate than in low-grade lymphomas (17%). None of the five high-grade lymphomas showed DNA aneuploidy. The chromosome study was successful in 81% of cases (39 of 48), and clonal chromosome abnormalities were observed in 92% of these (36 of 39). In most of the chromosomally abnormal clones the chromosome number was in the diploid range. Most tumors with pseudodiploid (46 chromosomes), hypodiploid (45-44 chromosomes), or hyperdiploid (47-49 chromosomes) clones were DNA diploid by flow cytometry. On the other hand, all specimens with a chromosome number exceeding 50 were DNA aneuploid by flow cytometry. Therefore, flow cytometric DNA analysis appears to be a rather coarse method that will detect aneuploidy only when there is a major increase in chromosome material.

Anti-melanoma antibodies bind preferentially to diploid metastases in immunoscintigraphy.

Immunoscintigraphy with 99Tcm-labelled anti-melanoma monoclonal antibody F(ab')2- fragments was performed in 23 patients with histologically verified metastatic melanoma. Immunoscintigraphy was positive in 14 patients and all known metastases were detected in eight patients, five of whom had only one lesion. Lesion localization and detectability were as follows: 12/13 (92%) cutaneous and subcutaneous, 11/14 (79%) lymph node, 5/7 (71%) bone, 3/6 (50%) lung and 1/5 (20%) abdominal metastases were visualized. Despite its high specificity--no false positive immunoscintigrams--the low sensitivity of this method in detecting deep metastases hampers its usability. The false negative results were not due to lack of antigen expression as positive immunostaining results were observed also in specimens from patients with negative immunoscintigrams. Flow cytometric analysis of the metastases revealed that in 7/8 (88%) patients with diploid tumours had positive immunoscintigrams but only 7/15 (47%) patients with aneuploid tumours. These results show that the diagnostic accuracy of melanoma immunoscintigraphy can be improved by selecting patients not only by testing for the antigen but also on the basis of DNA analysis of an accessible lesion.

Diploid predominance in hereditary nonpolyposis colorectal carcinoma evaluated by flow cytometry.

Fifty-nine colorectal carcinomas of patients with verified cancer family syndrome (CFS) were analyzed for DNA ploidy using flow cytometry. Sixty-eight percent of the tumors were diploid, and 32% were aneuploid. The aneuploid tumors had a median DNA index of 1.24 (range, 1.12-1.97). In 90% of all tumors the DNA index was less than 1.27. This predominance of diploid/near-diploid tumors was seen both in primary and in metachronous carcinomas. In 21 cases a cell cycle analysis was possible. Tumors with the S-phase fraction (SPF) greater than or equal to 9.8% had a worse prognosis than tumors with the SPF of less than 9.8%. These findings suggest that the predominance of diploid/near diploid DNA values is one of the characteristics of colorectal carcinomas in CFS. This might signify the existence of two or more pathogenetically different subgroups of colorectal carcinoma and explain the proposed better prognosis of colorectal carcinoma in CFS compared with other colorectal carcinomas.

Cell proliferation and expression of cytokeratin filaments in F9 embryonal carcinoma cells.

A double immunofluorescence method was developed for the monitoring of proliferation and differentiation of F9 embryonal carcinoma cells. Cytokeratin filament expression was used as a marker for differentiation, and proliferating cell nuclear antigen (PCNA)/cyclin or bromodeoxyuridine labeling were used as markers for proliferation. F9 cells had a high proliferation rate and were cytokeratin-filament-negative. Upon treatment with retinoic acid and dibutyryl cyclic AMP, cytokeratin-filament-positive cells with differentiated phenotype appeared. After 3 days, the extent of proliferation of cytokeratin-filament-positive cells was comparable to, but after 5 days significantly lower than, that of cytokeratin-filament-negative cells in the same culture. In differentiating F9 cells, cytokeratin filament expression is associated with, and even slightly precedes, the dramatic decrease in the rate of proliferation.

Teratocarcinoma stem cells as a model for differentiation in the mouse embryo.

Embryonal carcinoma (EC) cells, which are the malignant stem cells of teratocarcinomas, are considered similar to early embryo cells. The EC cells can be grown in vitro, and many of them can be experimentally induced to differentiate; upon differentiation, the cells become benign. Here we review some of the changes that take place in the cellular and molecular characteristics of murine F9 EC cells as they differentiate into endodermal cells. Upon differentiation of F9 cells, distinct changes occur in their cell surface molecules, cytoskeleton-associated proteins and cell adhesion properties. Simultaneously, the rate of cell proliferation decreases due to a dramatic increase in duration of G1 and S phases of the cell cycle. The changes in gene expression and cell behavior occurring during endodermal differentiation of EC cells closely resemble those occurring when the endoderm differentiates in the embryo. Teratocarcinoma stem cell lines may thus be exploited to enhance understanding of both teratoma-type neoplasms and embryonic development.

Growth of human colorectal carcinoma implants in the mouse subrenal capsule assay.

The subrenal capsule assay (SRCA) in normal immunocompetent mice was performed from 1331 implants of 43 human colorectal carcinomas to evaluate the possible applications for clinical chemosensitivity testing. Also the effect of an immunosuppressive agent, cyclosporine, was tested on the growth of tumours. Histologically in all except one of 23 saline-treated tumours the original tumour tissue was replaced by granulation tissue and inflammatory cells. This was also true in cyclosporine-treated mice since in only one of the nine tests tumour cells were observed. The macroscopic growth of the implants in the cyclosporine-treated mice was significantly less than in the saline-treated mice. Flow cytometric DNA-analysis revealed that the difference between macroscopic growth of saline and cyclosporine-treated groups was observed only in DNA-diploid tumours. We conclude that new methods are required to preserve the viability of human colorectal carcinoma in the SRCA.

Periodate-lysine-paraformaldehyde as fixative for the study of duodenal mucosa. Morphologic and immunohistochemical results at light and electron microscopic levels.

Methods are described which utilize periodate-lysine-paraformaldehyde (PLP) as the only fixative for the investigation of nonfrozen small-bowel biopsy specimens by morphologic and immunohistochemical studies at both light and electron microscopic levels. Small-bowel biopsy specimens were obtained from children suspected of coeliac disease. In light microscopy, there was good morphology and haematoxylineosin staining of the mucosa. Immunofluorescence and immunoperoxidase staining of paraffin-embedded specimens enabled an accurate demonstration of immunoglobulins (IgA, IgM, IgG) in the duodenal mucosa. In conventional electron microscopy, PLP gave a reasonable preservation of ultrastructure. In immunoelectron microscopy, a pre-embedding immunoperoxidase technique provided exact ultrastructural localization of immunoglobulins. The methods are easy to adapt and they give reliable light and electron microscopic results.

Immunoelectron microscopic localization of immune deposits in IgA glomerulonephritis.

Renal biopsy specimens obtained from twelve patients with IgA glomerulonephritis (IgA GN) were studied by immunoelectron microscopy (IEM) concomitantly with light microscopy (LM), immunofluorescence microscopy (IF), and electron microscopy (EM). For IEM, we used horseradish peroxidase (HRP)-conjugated antisera to human immunoglobulins (Ig) and to the complement component C3, and a diffusion technique with periodate-lysine-paraformaldehyde (PLP)-fixed tissue-chopper or cryostat sections. Due to well-preserved ultrastructure and good penetration of the antisera in the tissue-chopper sections, a detailed analysis of the distribution of immune material in the glomeruli was possible. In cryostat sections, ice crystal artifacts could not be avoided. The typical features of IgA GN could be reliably confirmed by IEM. Furthermore, IEM revealed the presence of Ig's and C3 in mesangial channels, in the intracellular vacuoles of glomerular cells, and in the electron-lucent areas along the glomerular basement membranes (GBMs). Staining of the mesangial channels indicates that they represent a route for the immune material gaining access into the mesangium. Intracellular vacuoles suggest that the deposited immune material can be partly eliminated through endocytosis by glomerular cells. The presence of Ig's and C3 in the electron-lucent areas supplies an explanation to the discrepancy sometimes observed between a positive finding in IF and a lack of deposits in EM.