Shigeya Uejima

A Phase I Trial of Vaccination of CA9-Derived Peptides for HLA-A24-Positive Patients with Cytokine-Refractory Metastatic Renal Cell Carcinoma

Purpose: A phase I peptide vaccination trial was done in patients with progressive cytokine-refractory metastatic renal cell carcinoma (RCC) to assess both the toxicity and capability to induce immune responses of three peptides (CA9p219-227, p288-296, and p323-331) derived from CA9, a tumor-associated antigen ubiquitously expressed in RCC. Experimental Design: Twenty-three patients positive for human leukocyte antigen (HLA)-A24 with histologically confirmed RCC were enrolled. Eligibility included progressive disease after standard cytokine therapy with interleukin-2 and/or IFN-α. Patients were vaccinated s.c. with the three peptides emulsified in incomplete Freunds adjuvant at 2-week intervals. Pre- and post-vaccination blood samples were obtained for toxicity assessment and immunologic studies. Patients were monitored for clinical responses on a 3-monthly basis. Results: Vaccinations were well tolerated without any major adverse event. Most of the patients developed peptide-specific CTLs and/or immunoglobulin G reactive to the peptides after the 6th or 9th vaccination, followed by a gradual increase in both CTL frequency and levels of peptide-reactive serum IgG. Three patients with multiple lung metastases showed partial responses with disappearance and shrinking of metastatic lesions. Additionally, stable disease for >6 months was observed in six patients (median duration, 12.2 months). Moreover, the median survival time of all patients who were progressive at trial enrollment after failing immunotherapy was 21.0 months (5-35 months). Conclusions: These results suggest that vaccination of these peptides is safe and recommended for further trials for HLA-A24-positive metastatic RCC patients.

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.

Immunohistochemical Detection of Tissue-Infiltrating Lymphocytes in Bladder Tumors

Local immunocompetence in patients with bladder tumors was evaluated immunohistochemically using monoclonal antibodies. As well as investigating the subsets of peripheral blood lymphocytes by flow cytometry, the subpopulations of tissue-infiltrating lymphocytes were investigated in frozen specimens from bladder cancers (n = 25) and from noncancerous tissues (n = 4). Six serial sections were stained with Leu4, Leu7, Leu10, LeuM3, OKT4, and OKT8 antibodies, respectively. Marked lymphocytic infiltration into bladder tumors was a favorable prognostic sign. T cell infiltration was marked in the noninvasive group and was prominent around tumor cells, whereas B cells were rare in the stroma. T cells predominated over B cells in 19 of 25 bladder tumors. In patients with low-stage tumors, OKT8 cells were more prominent than OKT4 cells. NK cells accumulated around the cancer cells and infiltrated within cancer nests; NK cell infiltration was scanty in high-stage cases. There was a slight scattering of macrophages in 13 cases. In contrast to the findings in subsets of tissue-infiltrating lymphocytes, subsets of peripheral blood lymphocytes did not correlate with stage of disease. These findings suggest the presence of local immunosurveillance against bladder tumors. To elucidate host immune response against tumor, it may be important to characterize tissue-infiltrating lymphocytes.

Immunological evaluation of personalized peptide vaccination monotherapy in patients with castration-resistant prostate cancer.

(Cancer Sci 2010; 101: 601–608)

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.

Detection of ABH Antigens via Exfoliated Urinary Cells

The expression of ABH blood group antigens of bladder carcinomas heralds a relatively benign clinical course, whereas the antigens deletion in tumors is often predictive of tumor recurrence and invasion. Using exfoliated urinary cells, we tried the prospective detection of ABH antigens. Though interpretations were independent, the status of antigens on exfoliated cells accorded with that on tissue sections. Detection of blood group antigens via exfoliated urinary cells is a noninvasive, reliable test that could be used for screening and surveillance of patients with bladder tumors. This method permits monitoring of patient ABH isoantigens by prospective screening.