Mean Labeling Index Research Articles

Objective measurement of the different collagen types in the corpus cavernosum of potent and impotent men: an immunohistochemical staining with computerized-image analysis.

Quantitative measurements of the collagen types (I, II, and IV) in the corpora cavernosa of potent and impotent men were carried out to investigate whether quantitative immunohistochemistry might contribute additional information as to the cause of erectile dysfunction. The study group consisted of 22 men with various etiologies of impotence and 4 normal, potent men. The quantitative immunohistochemistry measurements were performed by means of a cell-image processor. Three variables for each of the three types of collagen were studied, namely, the mean optical density (MOD), which relates to histochemical staining intensity; the labeling index (LI), which is positively related to the percentage of immunostaining; and the quick score (QS) index, which takes into account both LI and MOD values. None of the quantitative parameters taken individually (monovariate statistical analyses) made it possible to obtain any statistically significant difference between the types of collagen of the group under study. The mean QS value recorded for collagen type IV was significantly lower than that noted for collagen type I in the psychogenic (P = 0.019), arteriogenic (P = 0.012), and venogenic (P = 0.001) groups, whereas the MOD value was significantly lower in the normal (P = 0.043), arteriogenic (P = 0.013), and venogenic (P = 0.001) groups but not in the psycogenic group. The mean MOD of collagen type III was intermediate between that of the other types. In contrast, the mean LI value recorded for collagen type IV was significantly lower only in the venogenic (P = 0.032) and psychogenic (P = 0.049) groups as compared with the other groups. No objective qualitative change in the collagen types was observed that could be correlated to the etiology of erectile dysfunction. The significant difference seen in the quantitative parameters with regard to collagen type IV and the observed increase in the type I/III collagen ratio might attest to the notion that the response of the erectile tissue to ischemia is similar to that of other organs. The net effect of these changes is a restricted capacity for corporal expansion and alteration of the veno-occlusive mechanism.

Expression and prognostic value of proliferating cell nuclear antigen in transitional cell carcinoma of the urinary bladder.

Forty-eight patients with transitional cell carcinima (TCC) of the bladder were investigated. Routine paraffin-embedded sections were stained with proliferating cell nuclear antigen (PCNA) monoclonal antibody in order to determine the growth fraction of the bladder tumors and to correlate this with tumor grade, stage, development of recurrence and survival rate during follow-up. PCNA positive staining was detected in 95.8% (46/48) of the tumors. The mean labeling index (LI) of superficial tumors (Ta-1, n = 28) was 12.58 +/- 12.33%, and 34.55 +/- 21.89% in invasive tumors (T2-4, n = 18). A similar correlation was found in association with tumor grade. The patients were followed up for a mean of 4.9 years (range 1-14 years). The mean PCNA LI in nonrecurrent (n = 21) and simple recurrent (n = 7) superficial tumors was 11.29 +/- 11.79% and 16.44 +/- 14.05%, respectively, the difference not being statistically significant. To access survival, tumors with a PCNA LI above and below the median level (21%) were compared. Those patients (n = 19) with an index of > 21% (the mean of all the PCNA values) had a worse prognosis than those (n = 27) with an index of < 21%, a difference which is statistically significant. These results suggest that PCNA LI in bladder cancer may prove to be an objective and quantitative assay of biological aggressiveness and provide significant prognostic information, although it does not help the selection of patients at risk of simple recurrence in superficial tumors.

The significance of blood group-related antigen A in a pancreatic cell line (PGHAM-1) in hamsters. With special reference to cell proliferation

The relationship between blood group-related antigen A and cell proliferation was studied in a pancreatic carcinoma cell line (PGHAM-1) induced by N-nitrosobis (2-oxopropyl) amine (BOP) in hamsters. In vitro, the cell proliferation of PGHAM-1 was inhibited in a culture medium with an added monoclonal antibody (MoAb) against blood group-related antigen A depending on its concentration. In in vivo experiments, immunohistochemical double staining was performed using MoAb against blood group-related antigen A and bromodeoxyuridine (BrdU) in pancreatic carcinomas by intrapancreatic transplantation of PGHAM-1. Antigen A was expressed in the cell membrane and cytoplasm of PGHAM-1 cells. The BrdU labeling index (LI) in lesions with strong expression of MoAb A (> or = 50%) was 27.4 +/- 5.00, whereas the BrdU LI in lesions with weak expression of MoAb A (< or = 10%) was 11.9 +/- 2.10. The mean LIs in strongly expressed lesions of MoAb A were higher than those in weakly expressed lesions of MoAb A (p < 0.01). These results indicated that antigen A might be associated with cell proliferation in pancreatic carcinomas induced in hamsters.

Neurotrophin receptor and tyrosine hydroxylase gene expression in aged sympathetic neurons

Ribonuclease protection measurements revealed decreases of 26% in p75 neurotrophin receptor mRNA and 30% in trkA mRNA in superior cervical ganglia (SCG) of aged Long-Evans rats. These declines were not related to the presence of a spatial memory impairment, whose presence is known to strongly predict increased hypothalamic-pituitary-adrenal axis activity in these aged animals. A similar decrease with age was observed in p75, but not cyclophilin mRNA levels in SCG from F-344 inbred rats. In situ hybridization with paired sections from mature and aged F-344 rats revealed a 25% decline in the mean neuronal labeling index (LI) for p75 mRNA. In other paired sections, mean trkA LI decreased 16%, tyrosine hydroxylase (TH) LI increased 74% and cyclophilin LI did not change. Neuronal hypertrophy, p75 decreases and TH increases all occurred to a greatest extent in intermediate-sized neurons, resembling those innervating the pineal and cerebral vessels. In contrast to other SCG targets, this innervation is known to decline nearly 50% with aging. Retrograde tracer/in situ hybridization studies will be required to establish whether decreased p75 represents a marker for selective axonal regression and also to determine the significance of increased TH and neuronal hypertrophy.

No prediction of recurrence of meningiomas by PCNA and Ki-67 immunohistochemistry.

Immunohistochemistry for the expression of the proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 was studied in 16 non recurring meningiomas, 11 meningiomas recurring as benign tumors, 6 recurring as atypical meningiomas and in 9 recurring as malignant meningiomas. Non recurring meningiomas were defined in this study as tumors without recurrence at least 8 years after surgery. In addition 16 benign recurrences, 14 atypical- and 12 malignant meningiomas were studied. In each group great variation of labeling indices (LI) = per cent of tumor cells labeled was observed, especially of PCNA LIs. The non recurring meningiomas displayed lower mean LI for PCNA and Ki-67 than did the recurring meningiomas of all groups but the differences were not statistically significant. The same pattern was seen when totally resected tumors were studied alone. Benign-, atypical-, and malignant meningiomas had labeling indices that were related to the grade of malignancy. Only PCNA LIs of atypical- and malignant meningiomas were statistically significantly higher than PCNA LIs of non recurring meningiomas. The study indicates that PCNA and Ki-67 are of minor value as predictors of recurrence of benign meningiomas.

Cell proliferation and regulation of negative growth factors in mouse liver foci.

Foci of altered hepatocytes are preneoplastic lesions capable of progressing to hepatocellular carcinomas. To characterize the growth of preneoplastic hepatic lesions, size of hepatic foci was analyzed with regard to growth factor regulation and hepatocyte proliferation in focal and non-focal hepatocytes. Twelve-day-old female B6C3F1 mice were initiated with a single dose of the potent mutagen N-nitrosodiethylamine (DEN) (5 mg/kg body weight). Beginning at 6 weeks of age, mice were exposed for 16 weeks to 2038 p.p.m. unleaded gasoline (UG) vapor or 1 p.p.m. ethinyl estradiol (EE) in the diet. Analysis of hepatic foci demonstrated that UG significantly increased, but EE significantly decreased the size of DEN-initiated foci. Hepatic labeling index (LI), as measured by the incorporation of 5-bromo-2'-deoxyuridine, was similar in non-focal hepatocytes at 16 weeks in all groups (0.4-0.8%) and greatly increased in hepatic foci. Hepatocyte LI was significantly increased in DEN/UG foci (29%, n = 41) and significantly decreased in DEN/EE foci (6%, n = 23) relative to DEN/control focal hepatocytes (18%, n = 25). The mean LI of foci correlated with the focal size differences observed in the treatment groups. Immunohistochemical analysis with antibodies directed to the negative growth regulator transforming growth factor-beta 1 (TGF-beta1) demonstrated a consistent decrease of TGF-beta 1 in DEN/Ct and DEN/UG hepatic foci relative to non-lesion hepatocytes. Similar results were seen with mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-II R), which facilitates activation of latent TGF-beta 1. In contrast, only 50% of DEN/EE foci had decreased levels of TGF-beta 1 and M6P/IGF-II R relative to non-focal hepatocytes. These data suggest that proliferative responses observed in hepatic foci may be correlated with foci size. In contrast, chemically induced proliferative responses in non-focal hepatocytes after subchronic exposure cannot necessarily be used to predict proliferative effects in preneoplastic cell populations. Furthermore, these studies suggest that hepatic foci may occur by M6P/IGF-II R enhancing activation of latent TGF-beta 1 in non-focal hepatocytes but not in the focal hepatocytes, thereby affording focal hepatocytes a selective growth advantage.

Cyclin D1 in astrocytic tumours: an immunohistochemical study.

Forty-eight astrocytic tumours were stained immunohistochemically with antibodies to the cell cycle-regulating protein, cyclin D1, and to the proliferation marker MIB1 (Ki-67) using formalin fixed paraffin embedded tissue and a microwave antigen retrieval system. Cases were classified by the WHO system (1993). The labelling indices (LI) for both antibodies were compared with each other and with the tumour type. The mean labelling indices for both antibodies increased with the degree of malignancy, and a significant difference was seen between the pilocytic astrocytoma and diffuse astrocytoma together vs anaplastic astrocytoma and glioblastoma together. However, within each tumour type there was considerable variation in the labelling indices and a clear cut off value could not be demonstrated. There was a strong positive correlation between labelling indices for cyclin D1 and MIB1 in diffuse astrocytoma, but this correlation broke down increasingly in anaplastic astrocytoma and glioblastoma. There was poor correlation between cyclin D1 and MIB1 in pilocytic astrocytoma, a feature which appeared to separate them from the diffuse astrocytoma. Average labelling indices for cyclin D1 were higher than those of MIB1, which suggests that cyclin D1 positive cells represent a pool of cells from which proliferation and hence MIB1 expression can take place. In conclusion, cyclin D1 is overexpressed in astrocytic tumours, more so with increasing grade of malignancy and in a way which approximately correlates with MIB1 expression.

Effect of hyperthermia 42.5°C/120 min on 3H-thymidine incorporation in different tissue components of Wilms' tumors: An in vitro study

Survival rates of Wilms' tumors are correlated to tumor histology. Clinical studies and histological investigations have shown that different histological tumor components of Wilms' tumors also reveal different sensitivities to cytostatic agents and ionizing radiation. The aim of this study is to examine the effect of hyperthermia to Wilms' tumors, generally, and to the different tumor tissues composing nephroblastomas. The 3H-thymidine labelling indices (LI) of 23 Wilms' tumors and one renal rhabdomyosarcoma were studied by an autoradiographic in vitro method at temperatures of 37.5 degrees C/120 min and 42.5 degrees C/120 min. The LI of each tumor was measured and likewise the LI of all histological tumor components defining standard risk and high risk. The effect of hyperthermia was calculated as the percentage of inhibition of 3H-thymidine incorporation. Labelling indices between 22.4% and 46.3% (mean 33.2%) characterized Wilms' tumors as highly malignant and fast growing tumors. The LI of Wilms' tumors of standard risk and high risk did not differ significantly (33.8% vs. 30.4%). Also, epithelium and blastema of the same groups showed comparable high LI of 31.4% and 34.4%, respectively, and of 32.1% and 26.8%, respectively. The LI of stroma was significantly lower (11.9% and 10.9%). The mean LI of fetal-like rhabdomyoblastic cell elements of ten tumors was 24.5%. These tumor cells revealed a significantly higher LI than rhabdomyosarcomatous cells in one Wilms' tumor and one renal rhabdomyosarcoma (10.2% and 9.0%, respectively). The LI of anaplastic structures of one tumor was more than twice as high as the LI of surrounding tumor tissue. In vitro hyperthermia significantly inhibited 3H-thymidine incorporation into all nephroblastomas. Inhibition ranged between 13.7% and 84.6%, at an average of 47.3%. Four high risk tumors, as well as the single anaplastic and rhabdomyosarcomatous cells responded significantly stronger to heat than standard risk tumors (mean inhibition of 62.6% vs. 42.1%). Hyperthermia was more effective for blastema compared to epithelium and stroma. There was no correlation between the effect of hyperthermia and the amount of the 3H-thymidine LI at normothermia. The tremendous inhibition of DNA synthesis by hyperthermia particularly in high risk tumors and highly malignant tumor components is of clinical interest for children with Wilms' tumors resistant to conventional therapy.

The labelling index of primitive plasma cells determines the clinical behaviour of patients with myelomatosis.

For patients with multiple myeloma the most important laboratory correlate of prognosis and disease activity is the bromodeoxyuridine (BrdUrd) plasma cell labelling index (LI). However, the traditional immunofluorescent microscope LI technique, like other manual enumeration assays, can suffer from poor precision and accuracy. In this study the LI of different subpopulations of plasma cells (CD38++) as determined by flow cytometry was correlated with disease state. The mean LI of the total CD38++ population was significantly higher (2.7 +/- 0.4%) than the LI determined by the traditional slide technique (0.6 +/- 0.1%) for 65 samples tested. Primitive plasma cells (CD38++, CD45++) had a higher labelling index than mature plasma cells (CD38++, CD45-) (7.0 +/- 1.3% v 1.8% +/- 0.3%) and in one patient the LI of the primitive plasma cells was 46%. In addition, the LI of the mature plasma cells was lower than the total plasma cell population. As expected, there was a significant difference between the LI of patients in plateau phase and progressive disease but this difference was greatest when the LI of the primitive plasma cells was studied (9.2 +/- 2.9% v 2.2 +/- 0.7%; z = 19.9, P < 0.001). This study has raised some concerns about the sensitivity and accuracy of the traditional labelling index and has shown that the increased LI associated with progressive disease is almost entirely attributable to an increase in the LI of the primitive plasma cell subpopulation and that the LI of primitive plasma cells provides a more clinically significant correlation with disease status than the traditional assay.

Potential doubling time and clinical outcome in head and neck squamous cell carcinoma treated with 70 GY in 7 weeks

Purpose : To study the predictive value of pretreatment potential doubling time and labeling index, as measured by flow cytometry in patients with head and neck squamous cell carcinoma treated with conventional radiotherapy. Methods and Materials : 70 patients with a squamous cell carcinoma of the oropharynx and 4 patients with another involved head and neck site were entered in this prospective study. The duration of the S phase (TS), the labeling index (LI), and the potential doubling time (T pot) were obtained by flow cytometry measurements of a tumor biopsy obtained after i.v. injection of 200 mg bromodeoxyyuridine to the patient. The treatment consisted of 70 Gy in 7 weeks, 2 Gy per fraction and five fractions per week. Results : The mean and median LI were 7.7% (standard deviation, SD: 5.0) and 6.3%, respectively. The mean and median TS were 9.3 h (SD: 3.6) and 8.3 h, respectively. The mean and median T pot were 5.6 days (SD: 5.4) and 4.6 days, respectively. No significant relationship was found between the T pot or LI and the tumor stage (T), nodal status (N), histological grade, and the site of the primary within the oropharynx. The only parameter significant associated with an increased risk of local relapse was the tumor stage ( p < 0.001). The mean T pot for the group of tumors that relapsed locally was 5.3 days (SD: 3.3), compared to 6.1 days (SD: 4.08) for those who did not relapse locally (NS). Two parameters were significantly associated with a decrease in disease-free (DFS) and overall survival, namely the tumor stage ( p < 0.005, and p < 0.001, respectively, for DFS and overall survival) and nodal involvement (p = 0.02 and (p > 0.005, respectively, for DFS and overall survival). The TS, LI, DNA index, and T pot were not significantly associated with local relapse, DFS, and survival, either in the univariate or in the multivariate analysis. Conclusions : The method used to evaluate tumor cell kinetics did not provide clinically relevant kinetic parameters for this type of cancer. The classic prognostic factors (tumor stage and nodal status) were strongly associated with clinical outcome.

Difference in glycohistochemical lectin staining of collagen fibers in the corpora cavernosa of normal and impotent men.

The objectives of this study were to investigate the value of glycohistochemical staining with three lectin types specific to a particular glycan structure (Arachis hypogaea [PNA], Triticum vulgare [WGA], and concanavalin A [Con A]) as a method of defining possible changes in the collagen structure in the corpora cavernosa in potent and impotent men. The study group consisted of 4 normal potent men and 22 men with various etiologies of impotence. The quantitative histochemical measurements were performed by means of a cell image processor. Two variables for each of the three types of lectins were studied. These were the mean optical density (MOD), which relates to glycohistochemical staining intensity, and the labeling index (LI), which is positively related to the percentage of immunostaining. Only WGA staining made it possible to discriminate significantly between the normal and pathological groups under study. The two parameters (LI, MOD) were significantly higher in the case of WGA staining in the normal group (P = 0.004 and 0.013, respectively). In contrast, only the mean LI value, in the case of the psychogenic and venogenic patients, reached a level of statistical significance (P = 0.005 and 0.001, respectively), when it increased from PNA through WGA to Con A histochemical staining. The two variables (LI, MOD) changed markedly from PNA through WGA to Con A in the arteriogenic patients (P = 0.003 and P < 0.001, respectively). WGA is of diagnostic value in distinguishing between normal and abnormal collagen in the corpora cavernosa. The difference in the lectin staining of the other groups, particularly the arteriogenic group, may be attributed to alterations in the glycosylation of the procollagen that are probably due to changes in the partial pressure of oxygen (PO2) level, an important cofactor in normal glycosylation. WGA staining may therefore be used as a marker to distinguish true psychogenic patients from those with organic diseases. Moreover, it may be used as an additional parameter in selecting the best candidates for penile revascularization.

Cell Proliferation in Rat Forestomach Following Oral Administration of Styrene Oxide

A series of range-finding studies was conducted in a limited number of male F344 rats on the relation between cell proliferation and styrene oxide (SO) given as gavage doses in corn oil ranging from 550 to 1500 mg SO/kg. In each study, rats were injected with [3H]thymidine (0.50 mCi/g, ip) at intervals from 1 to 48 hr after dosing with SO. One hour later, stomachs were removed and fixed in formalin. Autoradiograms were prepared and labeling index (LI) was determined as the percentage of epithelial cells with3H-labeled nuclei. Mean LI increased with a peak at ∼15 hr after one or nine doses of SO. The increases were multifocal and not restricted to the area near the limiting ridge. The magnitude of the response in LI at 24 hr after dosing tended to decrease with progressive multiple doses (3/week). Dose-related morphologic lesions from SO (particularly submucosal) were multifocal and variable across the forestomach; they appeared unrelated to LI in a given area. In a final study, groups of 10 rats were given a single dose of 0, 20, 50, 125, 250, 500, or 800 mg/kg and LI was determined 15 hr later. Mean LI was dose-related with increases up to 250 mg/kg. A maximum response had apparently been reached and higher doses did not cause any further increase in LI. The degree of involvement of cell proliferation in the tumorigenicity of SO remains uncertain; additional studies are suggested to help in the understanding of such a possible relation.

Cell Proliferation in Rat Forestomach Following Oral Administration of Styrene Oxide

A series of range-finding studies was conducted in a limited number of male F344 rats on the relation between cell proliferation and styrene oxide (SO) given as gavage doses in corn oil ranging from 550 to 1500 mg SO/kg. In each study, rats were injected with [3H](0.50 mCi/g, ip) at intervals from 1 to 48 hr after dosing with SO. One hour later, stomachs were removed and fixed in formalin. Autoradiograins were prepared and labeling index (LI) was determined as the percentage of epithelial cells with 3H-labeled nuclei. Mean LI increased with a peak at ˜ 15 hr after one or nine doses of SO. The increases were multifocal and not restricted to the area near the limiting ridge. The magnitude of the response in LI at 24 hr after dosing tended to decrease with progressive multiple doses (3/week). Dose-related morphologic lesions from SO (particularly submucosal) were multifocal and variable across the forestomach; they appeared unrelated to LI in a given area. In a final study, groups of 10 rats were given a single dose of 0, 20, 50, 125, 250, 500, or 800 mg/kg and LI was determined 15 hr later. Mean LI was dose-related with increases up to 250 mg/kg. A maximum response had apparently been reached and higher doses did not cause any further increase in LI. The degree of involvement of cell proliferation in the tumorigenicity of SO remains uncertain; additional studies are suggested to help in the understanding of such a possible relation.

Immunohistochemical evaluation of proliferating cell nuclear antigen, prostate‐specific antigen and α1‐antichymotrypsin in human prostate cancer

To determine the relationship between growth fractions defined by proliferating cell nuclear antigen (PCNA), prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) staining in prostate cancer. A total of 96 lesions, including 71 from prostate cancers and 25 from benign prostatic hyperplasia (BPH) were evaluated in microscopic sections of the prostatic tissues from 34 patients with prostate cancer. Immunohistochemical staining was performed with an avidin-biotin system using monoclonal anti-PCNA antibodies, polyclonal anti-PSA and anti-ACT antibodies. There was a significant difference in the mean PCNA labelling index between tissue from prostate cancer (4.2 +/- 7.1) and BPH (0.5 +/- 1.1) (P = 0.002). The mean labelling index of PCNA tended to increase with increasing Gleason score. The proportion of cells positive for PSA was significantly higher in tissue from BPH than from prostate cancer (P = 0.005). While the proportion of cells immunostaining for ACT was significantly higher in tissue from BPH compared to that from prostate cancer (P = 0.02), there was no significant difference in the proportion of ACT-positive cells among prostate cancers of differing Gleason score. The mean labelling index of PCNA decreased significantly with the increase in the proportion of PSA-positive cells (P = 0.013). There was a significant relationship between the proportion of ACT- and PSA-positive cells (P = 0.001). These results indicate a reciprocal relationship between cell growth and tumour differentiation in prostate cancer. Although the significance of ACT deserves further study, there was evidence for the complexing of PSA with ACT from the immunohistochemical studies.

Immunohistochemical study of Fas antigen in liver of patients with chronic hepatitis and autoimmune liver disease

To examine the presence of Fas antigen in the liver in various chronic liver diseases, we stained Fas antigen immunohistochemically in the livers of 12 patients who were diagnosed as chronic hepatitis C in five, chronic hepatitis B in three, primary biliary cirrhosis in three, and autoimmune hepatitis in one. Three specimens with non-specific reactive hepatitis (NSRH) were used as histological controls. We prepared two kinds of rabbit polyclonal antibodies (anti-C, anti-D) as the primary antibody, raised against synthetic polypeptides from the two regions of human Fas antigen (C region, aa 300–314; D region, aa 104–114). The degree of the presence of Fas antigen expressed as the mean labeling index was relatively higher in chronic liver disease than in NSRH. Fas antigen was stained in the cytoplasm regardless of differences in primary antibody, but more diffusely in antibody C. Fas antigen was randomly distributed in the lobule and mainly concentrated in the periportal area associated with piecemeal necrosis. These results suggest that Fas antigen is related to liver injury from chronic viral hepatitis and autoimmune liver disease.

Heterogeneity of intratumour proliferative activity in primary breast cancer: Biological and clinical aspects

The present retrospective study was undertaken to verify whether the extent of intratumour proliferative activity variation or the method of quantifying tumour proliferative activity is related to biological characteristics and clinical outcome in a series of operable node-negative breast cancer patients. For tumour proliferative activity evaluation, the 3H-thymidine autoradiographic assay was used. After incubation of 3–8 samples from different areas of the equatorial section of each tumour for 1 h at 37°C with 3H-thymidine, the following methods were used for evaluation of tumour cell labelling: mean tumour labelling index (LI), the highest labelling value from a specific area (LI-max), and the extent of intratumour labelling variation from several samples (LI-CV). LI-max was related to ER and PgR status, and linearly correlated with LI (c.c. = 0.92, P < 10 −6) whereas LI-CV was independent of tumour size, grade ER and PgR status, but dependent on the number of tumour samples analysed for each tumour. After 5 years of median follow-up, disease-free survival was only related to tumour size (T1 versus T2: 84 versus 64%, P < 0.04 by log rank analysis) and different LI values (low versus high 3H-Tdr-LI: 86 versus 61%, P < 0.03 by log rank analysis). LI-max and LI-CV values were not significantly related to clinical outcome. Cox multivariate analysis confirmed the independent prognostic value of LI and tumour size on disease-free survival.

Cellular proliferation in prostatic adenocarcinoma as assessed by bromodeoxyuridine uptake and Ki‐67 and PCNA expression

Prostatic carcinomas vary in their biological potential, even when stratified by grade and stage. Measurement of cellular proliferation by various methods has been shown to correlate with outcome for several human cancers, including prostatic carcinoma. Uptake of bromodeoxyuridine (BrdUrd), a thymidine analogue, has been accepted as a measure of cellular proliferative rate. However, the technique is somewhat complex, requiring incubation with fresh tissue. We compared cellular proliferation as measured by BrdUrd uptake with two more simple immunohistochemical methods in 44 prostatic adenocarcinoma specimens and correlated the results with standard clinical parameters. The tissue was obtained via needle biopsy, channel transurethral resection, and radical prostatectomy. Specimens were incubated in vitro with BrdUrd and then fixed and paraffin embedded. Sections were immunohistochemically stained with antibodies to BrdUrd, proliferating cell nuclear antigen (PCNA), and Ki-67. At least 1,000 cells were scored, and a labeling index (LI) was calculated (number of positive cells/total number of cells). The mean LI determined by all three indices was low (BrdUrd = 3.0, PCNA = 7.0, Ki-67 = 3.4), consistent with the knowledge that prostatic tumors grow slowly. In 36 patients who had not been treated at the time of analysis, the LI as determined by all three methods correlated well with clinical stage and pathological grade. Furthermore, the LIs discriminated between those with tumor confined to the prostate and those with extension to the seminal vesicles, lymph nodes, or bone (P = 0.003, 0.004, 0.008 for BrdUrd, PCNA, and Ki-67, respectively). The LIs for PCNA and Ki-67 correlated well with that for BrdUrd (r = 0.84; r = 0.85), while the LIs for Ki-67 and PCNA correlated slightly less well with each other (r = 0.78). PCNA and Ki-67 expression appear to reflect essentially the same biological process as BrdUrd uptake. Either can substitute for BrdUrd as a measure of cellular proliferation, and Ki-67 seems to offer the fewest technical problems.

Microvessel density, endothelial cell proliferation and tumour cell proliferation in human colorectal adenocarcinomas

Thymidine incorporation studies performed in animal tumour models, revealed major differences in endothelial cell proliferation when tumour tissue was compared with normal tissue. The fraction of proliferating endothelial cells is reported to be increased by a factor of 30 to 40 in tumour tissue. To make it possible to analyze the endothelial cell proliferation in human tumours, an immunohistochemical double staining technique comprising CD31, an endothelial cell marker, and Ki-67, a proliferation marker, was developed. Endothelial cell proliferation was analysed in 21 primary human colorectal adenocarcinomas and in the adjacent mucosa. Proliferating endothelial cells were found throughout the entire carcinoma. The mean overall endothelial cell labeling index (ECLI) was 9.9% (range, 5.4-18.0), and the labeling index of endothelial cells in areas of intense neovascularisation was even higher. Mean ECLI in the vascular hot spots was 21.0% (range, 6.8-35.0), and the mean tumour cell labeling index (TCLI) in the maximally Ki-67 immunostained areas was 78.3% (range 47.0-89.7). In 14 of 21 carcinomas, these areas were predominantly found at the luminal margin of the tumour, as were the vascular hot spots. A significant positive correlation was found between tumour vascularity, measured in the vascular hot spots, and tumour cell proliferation, measured in the maximally Ki-67 immunostained areas (p < 0.05). To analyse this relation in more detail, microvessel density (MVD), TCLI and ECLI were determined per x400 microscopic field by scanning in sequence from the luminal tumour margin to the invasive tumour base. In all tumours, the pattern of the MVD per x400 field, from the luminal margin to the tumour base, was similar to that of the TCLI and ECLI. These findings confirm that the fraction of cycling endothelial cells is higher in human colorectal carcinoma than in the adjacent mucosa which suggests that endothelial cells are proliferating in most of the individual capillaries in tumour tissue. Regional differences in MVD correlate with differences in tumour cell proliferation in these tumours.

Proliferating Cell Nuclear Antigen (PCNA) and p53 Protein Expression in Bowen's Disease

Bowen's disease is a premalignant dermatosis comprised of a clonal proliferation of atypical keratinocytes in the full thickness of the epidermis. To elucidate the relationship between the alteration of the p53 tumor suppressor protein and cell proliferation rate, we immunohistochemically examined the expression of proliferating cell nuclear antigen (PCNA) and p53 protein in 30 cases of Bowen's disease. All the cases exhibited the full-thickness distribution of PCNA-positive cycling cells in the lesional epidermis. Quantitation of PCNA staining by image cytometry revealed a mean labeling index (LI) of 75.1 +/- 20.3. p53 expression was detected in 13 cases (43%). Expression was diffuse (p53 LI > 50) in 9 cases, but focal (p53 LI < 30) in the other four. The mean PCNA LI of p53 diffusely positive cases was significantly greater than that of both p53 focally positive and p53 negative cases (89.3 +/- 10.1 vs 62.7 +/- 21.2, and 70.5 +/- 21.6; p < 0.01, respectively). These findings suggest that a high-level accumulation of p53 protein results in a more increased cell proliferation in Bowen's disease.

Cell kinetics of human ovarian cancer with in vivo administration of bromodeoxyuridine

Cell kinetics could have prognostic significance in human ovarian cancer and might also help in designing optimal therapy. No data are available on the in vivo kinetics of this tumor using bromodeoxyuridine (BrdU) infusion before surgery. The kinetic parameters of human ovarian carcinoma were investigated in vivo using BrdU incorporation and bivariate BrdU/DNA flow cytometric (FCM) analysis. Fifty-five previously untreated patients with ovarian cancer (F.I.G.O. clinical stage III and IV) were studied. BrdU (250 mg/sqm) was given i.v. 6 h before surgery and samples of primary tumor and metastasis biopsies were fixed in 70% ethanol. By coupling the BrdU immunoreaction with biparametric FCM analysis, the nuclear DNA content (i.e., ploidy status), the tumor labelling index (LI), the synthesis time (TS) and the potential doubling time of the tumor mass (Tpot) were obtained. BrdU immunodetection was done on histological sections of the same tumors. The majority of the tumors had a DNA aneuploid content (71.5%). The amount of BrdU-positive S-phase cells varied in different tumor samples and when several samples were taken from the same tumor. The proportion of BrdU-negative S-phase cells was large (50% of the total S phase cells) in almost all cases. The mean LI was 6.1% using FCM and 7.2% on visual count of the slide. The mean TS and Tpot were 14.7 h and 12.5 days, respectively. LI and TS were not correlated with clinical tumor stage, histological grading, residual tumor size or DNA ploidy, but Tpot was significantly higher (p < 0.05) in patients with residual tumor size < 2 cm. Univariate analysis showed that Tpot was significantly associated with the response after first-line chemotherapy (p < 0.009). In multivariate analysis only residual tumor size was related to the response. Although this in vivo BrdU technique provides information on the kinetic features of human ovarian cancers, it remains questionable whether this information has any additional value compared to currently used prognostic factors which are assessable clinically and surgically.