Hisao Matsuda

Cell Proliferation of Human Bladder Tumors Determined by Brdurd and Ki-67 Immunostaining

The cell proliferation of bladder tumors was assessed in situ using BrdUrd (bromodeoxyuridine) and Ki-67 immunostaining. BrdUrd is incorporated into S-phase cells, and Ki-67 monoclonal antibody recognizes a nuclear antigen present in proliferating, but not resting cells. The percentage of labeled cells was expressed as the labeling index (LI). The average BrdUrd LI obtained applying this method to normal epithelium and to transitional cell carcinoma were 4.1% and 13.1%, respectively, while the Ki-67 LI had average values of 6.2% and 17.8%, respectively. Labeled cells were distributed throughout both the basal and surface layers of transitional cell carcinoma. In general, the value of the BrdUrd LI was correlated to that of the Ki-67 LI. A relatively large fraction of labeled cells was found in high grade or invasive tumors. Bladder tumors with lymph node involvement had higher LI than those without. In addition, a high frequency of S-phase cells within tumor tissue appeared to indicate a great potential for malignancy and thus a poor prognosis. These findings indicate that immunostaining with BrdUrd and Ki-67 may be useful tools for easy and quick evaluation of proliferating cells in bladder tumors.

Immunocompetence of tissue infiltrating lymphocytes in bladder tumors.

Tissue infiltrating lymphocytes (TIL) in bladder tumors have been assumed to be an expression of local host resistance against the tumor. We investigated the functional activity of TIL compared to peripheral blood lymphocytes (PBL). Isolation of TIL was performed using the enzyme cocktail treatment with Ficoll-Hypaque discontinuous gradient centrifugation. Analysis of lymphocyte subsets by flow cytometry demonstrated Leu 4, 43.6% (T cells); Leu 10, 10.5% (B cells) and Leu 7, 13.1% (natural killer (NK) cells) in TIL. The cytotoxic activity of TIL and PBL was tested in a four hour 51Cr-release assay. Myeloid K562 cells (NK sensitive), HT 1197 (bladder tumor) and fresh bladder tumors were used as target cells. The spontaneous NK cell activity of PBL was 23.7%, whereas that of TIL was only 3.5%. However, in vitro culture with IL2 induced a significant augmentation of NK activity in TIL as well as in PBL. On the other hand, the spontaneous lymphokine activated killer cell (LAK) activity of PBL and TIL was very low. IL2-cultured PBL and TIL exhibited the highest levels of lysis against fresh bladder tumors. Unlike PBL, IL2-induced cytotoxicity of TIL against autologous bladder tumors was higher than that against allogenic bladder tumors. Immunomodulators OK432 and Il2 were injected intratumorally during endoscopy. Analysis of the lymphocyte subsets in TIL showed an increase of T and NK cells following immunomodulator injection. Endoscopic injection of immunomodulators into bladder tumors augmented NK cell functional activity in TIL as well as PBL. These findings suggest that local immunosurveillance is directed against bladder tumors. Further studies are required to understand more fully the local and systemic host immune responses in cancer.

Immunoresponse of Tissue Infiltrating Lymphocytes in Bladder Tumors

Local immunocompetence was evaluated immunohistochemically in patients with bladder tumors before and after local injections of an immunomodulator. The subpopulations of tissue infiltrating lymphocytes (TIL) were examined by staining six serial sections with Leu4, Leu7, Leu10, LeuM3, OKT4, and OKT8 antibodies. T cells predominated over B cells in 19 of 25 bladder tumors. T cell infiltration was prominent around tumor cells, and it was marked in non-invasive tumors. B cells were rare in the stroma. In patients with low-stage tumors, OKT8 cells were more prominent than OKT4 cells. NK cells accumulated within cancer nests but their infiltration was scanty in invasive bladder tumors. Before surgery, immunomodulators (OK-432, IL-2) were injected intratumorally. Their administration resulted in marked increase of T and NK cells, irrespective of the stage of disease; there was a slight increase in B cells. These findings suggest that local immunosurveillance plays a role against bladder tumors. Further studies are required to elucidate host immune responses in the microenvironment of the cancer site, as well as the systemic immune reaction.

Immunofluorescent study on the interaction between collagen and calcium oxalate crystals in the renal tubules

The interaction of calcium oxalate crystals and renal tubular cells was studied. Rat renal collecting tubular cells were cultured and immunologically stained with anti-type-IV collagen antiserum (type-IV collagen exists in renal tubular basement membrane). When renal tubular cells and calcium oxalate crystals were mixed, clumps were formed. These clumps were examined by immunological staining with anti-type-IV collagen antiserum. In another series of experiments, calcium-containing crystals were found to be adsorbed onto mucous threads and cast-like materials, although no such adsorption was observed on squamous cells. These absorbed materials interacted with anti-type-IV collagen antiserum. These results suggest that collagen in the renal tubular basement membrane may act as matrix in urinary stone formation.

Cell Proliferation of Human Bladder Tumors

The cell proliferation of bladder tumors was assessed via an immunohistochemical technique using anti-bromodeoxyuridine (BrdUrd) antibody. The incorporation of the thymidine analogue, BrdUrd, into DNA synthesis phase was performed by an in vitro labeling technique. The percentage of labeled S-phase cells was expressed as the labeling index (LI). Discrimination between BrdUrd-labeled and unlabeled cells was easy because of the absence of background staining. The average LI obtained using this method in normal, normal epithelium bearing bladder tumors and cystitis was 5.0%, 4.0% and 5.0%, respectively. On the other hand, LI in bladder tumors was 13.0%, while the difference from non-malignant bladder epithelium was significant. As in high grade tumors, the labeled cells distributed throughout both the basal and surface layers of epithelium, compared to low grade tumors and controls. However, there was some variation in the distribution pattern within the same tumor depending on the histological site. On the other hand, the higher S-phase fraction was found in high grade or invasive bladder tumors. Bladder tumors with lymph node involvement had higher LI than those without the involvement. In patients who underwent TUR, the LI was as high as 10.0% in the recurrent group compared with 7.3% in the non-recurrent group. Moreover, in the total cystourethrectomy group, higher LIs were obtained in a patient dying of cancer (20.0%) and a patient with postoperative metastasis (21.0%) than in the non-recurrent group (14.8%). The high frequency of S-phase cells within tumor tissue appears to indicate a more malignant potential and thus a poor prognosis. Although further studies are required for its full significance, the measurement of BrdUrd labeling index by in vitro labeling method promises to afford useful information regarding the biological behavior of bladder tumors.

The kinetics of cellular proliferation in rat urinary bladder treated with N-butyl-N-(4-hydroxybutyl)nitrosamine

The cellular proliferation of the bladder epithelium was determined sequentially in rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). The incorporation of bromodeoxyuridine (BrdUrd) into the DNA synthesis phase was determined by an in vitro labeling technique. The percentage of labeled cells was expressed as the labeling index (LI). The average LI values in normal subjects and cancer-bearing subjects were 5.1(%) and 24.2(%) respectively. With the transition of the bladder epithelium from simple hyperplasia to cancer, the LI values of the bladder epithelium were increased. Cases of papillary or nodular (PN) hyperplasia were divided into two types according to the localization of the BrdUrd-labeled cells. The LI in PN hyperplasia was 12.4(%); the difference from cancer was significant. In another experiment, the effect of partial cystectomy on bladder tumors was examined. The group with partial cystectomy showed increases in grade and stage of cancer, and the LI of cancer was more than that in the group without partial cystectomy. The results indicate that partial cystectomy may play a role as promotor for bladder tumors. The current study may be of more practical value than is the conventional one for investigating the histogenesis and progression of human bladder cancer.

Clinical assessment of carcinoma in situ of the human urinary bladder.

Recent interest in intravesical instillation of bacillus Calmette-Guérin for the management of carcinoma in situ (CIS) of the bladder prompted us to review our results of total immediate cystourethrectomy. From 1975 to 1987, we surgically treated 302 patients with primary bladder tumours. Of these, 21 bladder tumours were histologically diagnosed as CIS, and total immediate cystourethrectomy was performed in all cases. The lesions were classified into three types: primary CIS accompanied by neither previous nor simultaneous tumours of the urinary tract (Type 1,9 patients), concomitant CIS associated with superficial papillary tumour (Type 2, 6 patients), and secondary CIS detected during the follow-up period after treatment of the superficial papillary tumour (Type 3, 6 patients). Primary CIS was more often diagnosed according to subjective symptoms such as micturition patin than concomitant CIS. Secondary CIS was diagnosed in all patients by routine examinations including cytology and cystoscopy. There was no particular relationship between the time of recurence of the tumour and the occurrence of secondary CIS. Within the same period, 60 patients with stage T1 bladder tumour were treated by total cystourethrectomy. The actuarial 5-year survival rate was 77% for T1 and 75% for CIS. The 5-year survival rate was 71% for Type 1, 83% for Type 2, and 67% for Type 3 CIS with no difference among the CIS types.Tumour invasion to the bladder, prostate, ureter, or lymph nodes was observed in 9 (42.9%) of the 21 patients, although cystourethrectomy was performed within 3 months of the diagnosis. Examination of ABH antigens did not allow prediction of invasion of CIS.Our findings suggest that the invasive potential of CIS may seriously limit the indications of conservative treatment against carcinoma in situ.

Growth fraction of human bladder tumors

SummaryThe growth fraction of bladder tumors was immunohistochemically assessed in situ using anti-DNA polymerase (Pol α) monoclonal antibody. This enzyme is known to be present in the nucleus of the cells in G1, S, and G2 phases. The percentage of labeled cells was expressed as the labeling index (LI). The average LI was 6.0% in normal epithelium and 17.8% in bladder tumors, this difference being significant. The labeled cells were distributed throughout both the basal and surface layers of bladder tumors. However, in some bladder tumors, the distribution of Pol α-labeled cells varied from area to area. The higher fraction of labeled cells was found in high grade or invasive tumors. Papillary and nodular bladder tumors showed a greater rate of cell proliferation than papillary tumors. These findings suggest that Pol α immunostaining could be a potent tool for easy and quick evaluation of proliferating cells in bladder tumors, there-by providing a supplement to conventional histological findings.

ENHANCING EFFECT OF PARTIAL CYSTECTOMY ON RAT URINARY BLADDER CARCINOGENESIS

The effects of 5% and 50% partial cystectomies on bladder tumorigenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN) were investigated in Wistar rats by examining the histological findings and cell proliferative activity. The incorporation of bromodeoxyuridine (BrdUrd) into the DNA synthesis phase was determined by an in-vitro labeling technique. After eight weeks of treatment with drinking water containing 0.05% BBN, 5% or 50% partial cystectomy was performed at the end of week 16, and the resected bladder was sutured with dexon or silk. There was no difference in the incidence of papillary or nodular (PN) hyperplasia between the control and partial cystectomy groups. However, the incidence of cancer in the group given partial cystectomy was much higher than that in the control. All the cancers in the control group were grade-1 superficial tumors, whereas grade-2 or invasive tumor was observed in six of 40 animals in the partial cystectomy group. The 31.0% labeling index of cancer in the partial cystectomy group was greater than the 24.1% in the control group. There was also a significant difference in the number of BrdUrd-labeled cells in PN hyperplasia between the control and partial cystectomy groups. These findings indicate that partial cystectomy enhances BBN-initiated bladder carcinogenesis and the increase in DNA synthesis found in PN hyperplasia and cancer may be associated with the induction of bladder tumors.

Role of natural killer cells in bladder tumor.

The role of natural killer (NK) cells in bladder tumors was assessed from the aspect of local and systemic immune responses. The NK cell activity was measured in a 4-hour 51Cr-release assay. The NK activity in patients with bladder tumor was lower, though not significantly, than that in normal individuals. In patients with bladder tumor, the NK activity was significantly lower in invasive tumors and lymph node metastases. Moreover, the NK activity was lower in those who died (n = 4) than it was in survivors (n = 21). In an in vitro experiment, OK432 significantly augmented the NK activity in peripheral blood lymphocytes (PBL), however, this augmentation was not always OK432 dose-dependent. The augmented NK activity induced by OK432 occurred even in patients with invasive tumors. On the other hand, the spontaneous NK activity in tissue-infiltrating lymphocytes (TIL) and lymph node lymphocytes (LNL) was significantly lower than that in PBL. In these three groups, the NK activity was significantly increased by OK432, this rate of increase was highest in TIL, followed by LNL and PBL. Further studies are required to elucidate the role of NK cells in bladder tumor, from the aspect of local and systemic immune responses.