Expressions of PD-L1 and Nectin-4 in urothelial cancer patients treated with pembrolizumab

ObjectivesRecently, the standard of care for advanced urothelial cancer (UC) has been changed by developing immune-checkpoint inhibitors (ICIs). However, its response rate is limited to 20–30%. The identification of biomarkers to predict the therapeutic effects of ICIs is urgently needed. The present study explored the association between immunohistochemical biomarkers and clinical outcomes in UC patients treated with pembrolizumab.Patients and methodsA total of 85 patients with UC who received pembrolizumab after chemotherapy from January 2018 to May 2020 were retrospectively reviewed. Tumor tissues were obtained for immunohistochemical study from 47 out of 85 patients. The protein expressions of PD-L1, WT1, Nectin-4, CD4, CD8, Foxp3, and CD68 in tumor cells and/or tumor infiltrating lymphocytes were immunohistochemically examined. The associations between protein expressions and overall survival (OS), progression-free survival (PFS), and disease control rate (DCR) were statistically analyzed.ResultsPatients with positive PD-L1 in tumor cells showed significantly worse OS (Log-rank test: HR 5.146, p = 0.001, Cox regression analysis: HR 4.331, p = 0.014) and PFS (Log-rank test: HR 3.31. p = 0.022), along with significantly lower DCR (14.3%) compared to the PD-L1 negative patients (67.5%). In addition, patients with strong expression of Nectin-4 in tumor cells showed significantly higher DCR (100%) than the other patients (50%).ConclusionPD-L1 expression in tumor cells was associated with poor prognosis (OS and PFS) and low DCR. Interestingly, the strong expression of Nectin-4 was correlated with high DCR. PD-L1 and Nectin-4 expression in tumor cells could be prognostic biomarkers useful for pembrolizumab in patients with advanced UC.

Expressions of PD-L1 and Nectin-4 in urothelial cancer patients treated with pembrolizumab.

OBJECTIVES: Recently, the standard of care for advanced urothelial cancer (UC) has been changed by developing immune-checkpoint inhibitors (ICIs). However, its response rate is limited to 20-30%. The identification of biomarkers to predict the therapeutic effects of ICIs is urgently needed. The present study explored the association between immunohistochemical biomarkers and clinical outcomes in UC patients treated with pembrolizumab. PATIENTS AND METHODS: A total of 85 patients with UC who received pembrolizumab after chemotherapy from January 2018 to May 2020 were retrospectively reviewed. Tumor tissues were obtained for immunohistochemical study from 47 out of 85 patients. The protein expressions of PD-L1, WT1, Nectin-4, CD4, CD8, Foxp3, and CD68 in tumor cells and/or tumor infiltrating lymphocytes were immunohistochemically examined. The associations between protein expressions and overall survival (OS), progression-free survival (PFS), and disease control rate (DCR) were statistically analyzed. RESULTS: Patients with positive PD-L1 in tumor cells showed significantly worse OS (Log-rank test: HR 5.146, p = 0.001, Cox regression analysis: HR 4.331, p = 0.014) and PFS (Log-rank test: HR 3.31. p = 0.022), along with significantly lower DCR (14.3%) compared to the PD-L1 negative patients (67.5%). In addition, patients with strong expression of Nectin-4 in tumor cells showed significantly higher DCR (100%) than the other patients (50%). CONCLUSION: PD-L1 expression in tumor cells was associated with poor prognosis (OS and PFS) and low DCR. Interestingly, the strong expression of Nectin-4 was correlated with high DCR. PD-L1 and Nectin-4 expression in tumor cells could be prognostic biomarkers useful for pembrolizumab in patients with advanced UC.

Prognostic significance of tumor extension into venous system in patients undergoing surgical treatment for renal cell carcinoma with venous tumor thrombus.

AIMS: The objective of this study was to evaluate the prognostic significance of the extent of a tumor thrombus in renal cell carcinoma (RCC) involving the venous system. METHODS: This study included 135 consecutive RCC patients with a venous tumor thrombus undergoing radical nephrectomy and tumor thrombectomy between 1985 and 2009. These patients were classified based on the maximal level of the tumor thrombus extending into the venous system, as follows: group 1, renal vein; group 2, infradiaphragmatic; and group 3, supradiaphragmatic. RESULTS: Of the 135 patients, 65, 49 and 21 were classified into groups 1, 2 and 3, respectively. The 1, 3 and 5-year cancer-specific survival (CSS) rates in these 135 patients were 89.2, 56.9 and 49.2%, respectively. Among several factors examined, tumor size, tumor grade, perirenal fat invasion and presence of metastasis, but not extent of tumor thrombus, were significantly associated with CSS on univariate analysis. Of these significant factors, only tumor size and presence of metastasis appeared to be independently related to CSS on multivariate analysis. When the patients without metastasis were analyzed separately, CSS in groups 2 and 3 was significantly poorer than that in group 1. CONCLUSIONS: These findings suggest the absence of a significant prognostic impact of the level of the tumor thrombus in a complete cohort of RCC patients with a venous tumor thrombus; however, it is warranted to determine whether the level of the tumor thrombus has different effects on the prognosis according to the presence of metastatic diseases.

Enhanced sensitivity to androgen withdrawal due to overexpression of interleukin-6 in androgen-dependent human prostate cancer LNCaP cells.

BACKGROUND: The objective of this study was to investigate the effects of interleukin-6 (IL-6) overexpression in androgen-dependent prostate cancer LNCaP cells on their phenotype under an androgen-deprived condition. METHODS: We established IL-6-overexpressing LNCaP (LNCaP/IL-6) by introducing the expression vector containing IL-6 cDNA. Changes in the phenotype in LNCaP/IL-6 were compared with that in LNCaP transfected with control vector alone (LNCaP/Co). RESULTS: In vitro, the growth of LNCaP/IL-6 was significantly inferior to that of LNCaP/Co under an androgen-deprived condition. Similarly, LNCaP/IL-6 tumour in nude mice rapidly regressed after castration; however, LNCaP/Co tumour growth was transiently inhibited after castration and then continuously accelerated. After androgen withdrawal, expression levels of phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and Akt in LNCaP/IL-6 were markedly upregulated compared with those in LNCaP/Co; however, additional treatment with specific inhibitor of the MAPK or Akt signalling pathway significantly inhibited the growth of LNCaP/IL-6 compared with that of LNCaP/Co. Furthermore, gene microarray analyses showed that androgen deprivation resulted in differential expression of genes involved in growth, apoptotsis and tumorigenesis between LNCaP/Co and LNCaP/IL-6. CONCLUSION: Excessive secretion of IL-6 by LNCaP cells in an autocrine manner may have a suppressive function in their growth and acquisition of androgen-independent phenotype under an androgen-deprived condition.

Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis.

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.

Troglitazone enhances glucose uptake induced by alpha-adrenoceptor stimulation via phosphatidylinositol 3-kinase in rat heart.

1. Thiazolidinedione-derived agents have been reported to act as insulin sensitizers by augmenting insulin-dependent stimulation of phosphatidylinositol 3-kinase (PI3K) activity in a specific manner. It has been suggested that alpha-adrenoceptor stimulation mediates glucose uptake through PI3K in the heart. 2. To elucidate whether the thiazolidinedione-derived agent troglitazone (TRO) affects glucose uptake induced by alpha-adrenoceptor stimulation through PI3K, the rate of glucose uptake was quantified from the rate of accumulation of sugar phosphate (d[SP]/dt) using [(31)P] nuclear magnetic resonance spectroscopy after substitution of glucose with 2-deoxyglucose in rat perfused heart. Hearts were stimulated with 100 micromol/L phenylephrine plus 10 micromol/L propranolol (alpha-adrenoceptor stimulation), or 1 micromol/L isoproterenol plus 10 micromol/L phentolamine (beta-adrenoceptor stimulation). 3. The d[SP]/dt in the alpha- and beta-adrenoceptor-stimulated groups (0.45 +/- 0.06 and 0.42 +/- 0.04 micromol/min per g, respectively) was higher than that of the control group (0.27 +/- 0.02 micromol/min per g; P < 0.01). The addition of 2 microg/mL troglitazone to alpha-adrenoceptor stimulation augmented d[SP]/dt (0.72 +/- 0.08 micromol/min per g; P < 0.05 vs the alpha-adrenoceptor-stimulated group), which was effectively blocked by 3 micromol/L wortmannin (0.35 +/- 0.06 micromol/min per g; P < 0.01 vs troglitazone + alpha-adrenoceptor stimulation group). However, addition of troglitazone to beta-adrenoceptor stimulation did not alter d[SP]/dt (0.33 +/- 0.02 micromol/min per g; P = NS vs the beta-adrenoceptor-stimulated group). 4. These results indicate that troglitazone acutely enhances alpha-adrenoceptor stimulation on glucose uptake through a PI3K-dependent pathway, thus possibly improving glucose utilization in a catecholamine-released state.

Characterization of rice anthranilate synthase alpha-subunit genes OASA1 and OASA2. Tryptophan accumulation in transgenic rice expressing a feedback-insensitive mutant of OASA1.

Anthranilate synthase (AS) is a key enzyme in the synthesis of tryptophan (Trp), indole-3-acetic acid, and indole alkaloids. Two genes, OASA1 and OASA2, encoding AS alpha-subunits were isolated from a monocotyledonous plant, rice (Oryza sativa cv Nipponbare), and were characterized. A phylogenetic tree of AS alpha-subunits from various species revealed a close evolutionary relationship among OASA1 and Arabidopsis ASA2, Ruta graveolens AS alpha 2, and tobacco ASA2, whereas OASA2, Arabidopsis ASA1, and R. graveolens AS alpha 1 were more distantly related. OASA1 is expressed in all tissues tested, but the amount of its mRNA was greater in panicles than in leaves and roots. The abundance of OASA2 transcripts is similar among tissues and greater than that of OASA1 transcripts; furthermore, OASA2 expression was induced by a chitin heptamer, a potent elicitor, suggesting that OASA2 participates in secondary metabolism. Expression of wild-type OASA1 or OASA2 transgenes did not affect the Trp content of rice calli or plants. However, transformed calli and plants expressing a mutated OASA1 gene, OASA1(D323N), that encodes a protein in which aspartate-323 is replaced with asparagine manifested up to 180- and 35-fold increases, respectively, in Trp accumulation. These transgenic calli and plants were resistant to 300 microM 5-methyl-Trp, and AS activity of the calli showed a markedly reduced sensitivity to Trp. These results show that OASA1 is important in the regulation of free Trp concentration, and that mutation of OASA1 to render the encoded protein insensitive to feedback inhibition results in accumulation of Trp at high levels. The OASA1(D323N) transgene may prove useful for the generation of crops with an increased Trp content.

Small cell carcinoma of the kidney: case report.

An 83-year-old female diagnosed with small cell carcinoma of the kidney is reported on. The clinical picture was similar to that seen with transitional cell carcinoma of the renal pelvis. A right nephroureterectomy was performed and a histological examination revealed similar morphological features to those of small cell carcinoma of the lung. Neuroendocrine differentiation was proven by a positive immunoreaction to neuron-specific enolase. A review of the literature indicated that in the urinary tract, most of the cases of small cell carcinoma occurred in the urinary bladder, with this case being the eleventh reported case of small cell carcinoma originating in the kidney. As with many of the other cases of small cell carcinoma of the urinary tract, this patient's tumor was associated with both adenomatous and squamous differentiation. The patient died 2 months after surgery, prior to any chemotherapy administration.

A fungal chitinase gene fromRhizopus oligosporus confers antifungal activity to transgenic tobacco.

We have studied whether a chitinase involved in cell autolysis of a filamentous fungus,Rhizopus oligosporus, can operate as an antifungal defense system in tobacco. Thechi1 gene was introduced into tobacco by theAgrobacterium tumefaciens leaf disc system. Among 22 transgenic tobacco plants, 2 were selected and their individual homozygous progeny, Tch1-1 and Tch2-1, were studied. Chitinase activity in the extracts of young leaves from Tch1-1 or Tch2-1, in which thechi1 gene product was detected by Western blot analysis, was three- to four-fold higher than that from the control plants. A fungal infection assay on the leaves infected with the discomycete pathogensSclerotinia sclerotiorum andBotrytis cinerea revealed that the symptoms observed with these two were remarkably suppressed as compared with the control leaves.

Enhancement of an Mg(2+)-dependent nuclease activity in rat liver cells exposed to cisplatin.

An Mg(2+)-dependent deoxyribonuclease activity was highly purified from a 0.6 M NaCl extract of rat-liver nuclei. The template primer activity assay with a Klenow polymerase suggested that this nuclease plays a role in incision/excision of cisplatin-modified DNA strands to form single-strand gaps. In another experiment, rat-liver (Ac2F) cells were cultured in the presence of cisplatin. A 0.6 M NaCl extract was prepared from the cultured-cell nuclei and subjected to the activity blotting analysis [Seki et al. (1993) J. Chromatography 618, 147-166]. The nuclease activity of the extract was enhanced in response to cisplatin, but not in the presence of cycloheximide. These results imply that cisplatin-DNA lesions induce the Mg(2+)-dependent deoxyribonuclease activity in Ac2F cells to provide priming sites for the repair synthesis.

[A case of renal pseudotumor caused by pyelonephritis].

A case of renal pseudotumor due to pyelonephritis is reported. A 70-year-old female was admitted to our hospital for the evaluation of right renal mass in pyelogram. She had had episodes of pyelonephritis many times. Computed tomography (CT) revealed poorly marginated low density mass that exhibits inhomogenous enhancement with contrast medium. Angiography showed an avascular mass. On magnetic resonance imaging (MRI), the mass was isointensity or slightly low intensity on T1 weighted scan, while it showed low intensity on T2 weighted scan. The patient died from small cell carcinoma in the lung two years later. Autopsy was performed and no malignancy was found in the kidney. MRI was useful to differentiate such inflammatory change from renal tumor, which is mostly high intensity on T2 weighted scan.

[Congenital arteriovenous malformation of the kidney: report of two cases--detection by digital subtraction angiography with carbon dioxide].

Two cases of arteriovenous malformation of the kidney were reported. The first case was a 19-year-old female, complaining of right flank pain and gross hematuria. Right selective renal arteriography revealed a 2 x 1.5 cm large cisoid type arteriovenous malformation at the most distal region of the lower branch of the renal artery. Transcatheter embolization, using Gelfoam and absolute ethanol, was successfully done, which was confirmed with repeated digital subtraction angiography with carbon dioxide (CO2-DSA). The second patient was a 55-year-old female with past history of right idiopathic renal bleeding, complaining of right flank colicky pain and gross hematuria. Right selective renal arteriography was done without any pathological findings, while CO2-DSA documented an arteriovenous malformation in the hilar region. Transcatheter embolization was not done, because the malformation seemed to be proximally located. Thus CO2-DSA was thought to be a reliable method in the diagnosis of arteriovenous fistula.

[A case of retroperitoneal fibrosis with special emphasis on diagnosis using the magnetic resonance imaging].

A case of idiopathic retroperitoneal fibrosis (RF) is reported. MRI showed a low signal intensity on T1 and heterogeneous signal intensity on T2-predominant image. Although this could be demonstrated either in malignant RF or early stage of non-malignant RF, differentiation from malignancy was made from the quick response to steroid therapy.

[Complete testicular feminization syndrome associated with thermolabile androgen receptor].

Radioreceptor assay and thermostability test for the androgen receptor in two cases with complete testicular feminization syndrome were performed in regard to the fibroblasts cultured from genital skin on the basis of dispersed whole cell binding assay (Eil et al., 1980). No [3H]dihydrotestosterone binding to the androgen receptor was observed in case 1 (receptor negative), while maximum binding capacity and dissociation constant of androgen receptor for [3H]dihydrotestosterone in case 2 were 21000 sites per cell and 1.67 x 10(-10) M (receptor positive). The specific binding of [3H]dihydrotestosterone to the androgen receptor in case 2 decreased remarkably to 6.6% after high temperature (42 degrees C) incubation in comparison with that at 22 degrees C incubation. The specific binding of [3H]dihydrotestosterone to the androgen receptor in normal controls decreased down to 81.6% at high temperature incubation. Thermostability test was useful to demonstrate qualitative abnormality of androgen receptor in receptor positive testicular feminization syndrome.

Plant regeneration from protoplasts isolated from embryogenic suspension cultures of creeping bentgrass (Agrostis palustris Huds.).

A rapidly growing embryogenic suspension culture cell line of creeping bentgrass cv Penncross (Agrostis palustris Huds.) was established from callus derived from the culture of mature seeds. High concentrations of 2,4-D were required for the induction of callus (3 mg/1) as well as for the maintenance of the cell Une (2 mg/1) on modified B5 medium of Gamborg. Protoplasts isolated from the suspension cultures were successfully cultured in Murashige and Skoog's basal medium supplemented with only 0.1 mg/1 2,4-D. Although protoplast plating efficiency was rather low (0.36%), 30% of the protocalli formed normal green plants that were successfully established in soil.

Is hormonal therapy necessary in prepubertal boys with cryptorchidism?

Three hundred and twenty-eight prepubertal boys with cryptorchidism who had orchiopexy were reviewed in regard to the attachment of the gubernaculum. Normal attachment of the gubernaculum was found in only 19 out of 240 unilateral testes (8%), and in 12 out of 176 testes (7%) of 88 patients with bilateral cryptorchidism. Abnormal attachment of the gubernaculum was found in 307 out of 338 testes (91%). These data suggest that hormonal therapy for undescended testis may be effective in only 9% of the cases with cryptorchidism, and it cannot replace surgery.

Androgen receptor levels in patients with isolated hypospadias.

The binding of [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) to the androgen receptor was studied in 26 prepubertal boys with isolated hypospadias (no associated anomaly) using fibroblasts cultured from surgical specimens of hypospadiac penile skin and/or chordal tissue. Maximal binding capacities of androgen receptor for [3H] dihydrotestosterone in 10 patients with mild degree (2 glandular + 8 penile), 6 with severe degree (4 penoscrotal + 2 scrotal), chordal tissues of hypospadias (N = 10), and controls (10 endocrinologically normal boys) were, respectively, 11750 +/- 2410, 10720 +/- 2680, 16100 +/- 7570, and 11570 +/- 3830 (mean +/- sd) sites per cell. Dissociation constants in these subjects were 1.09 +/- 0.29, 0.93 +/- 0.22, 1.20 +/- 0.31, and 1.22 +/- 0.41 x 10(-10) mol/l respectively. There were no significant differences as to both maximal binding capacity and dissociation constant of androgen receptor among any groups. Abnormal receptor thermolability was not found in 5 patients with severe degree of hypospadias either. It is, therefore, concluded that prepubertal boys with isolated hypospadias have primarily normal androgen receptor activity.

Heating system with a lens applicator for 430 MHz microwave hyperthermia.

To attain deep and localized heating for hyperthermic treatment of cancers, metal plate lens applicators that can converge microwave electromagnetic (EM) energy in the lossy medium such as human muscle with a computer-controlled heating system have been developed. This paper describes a system operating at 430 MHz. Results of an electric-field distribution calculation and a heating experiment made on a saline solution phantom show that the maximum heating depth is over 60 mm using the lens applicator, which is twice as deep as that obtained with a conventional waveguide applicator. The experimental results agree well with the theoretical ones. A computer-controlled heating system has been developed using the applicator. Experimental results show that fluctuations in temperature at locations in the heating region of the saline phantom were maintained within +/- 0.3 degrees C of the present temperatures. These results indicate that the system can be used for the clinical hyperthermia treatment of cancer.

[Clinical statistics on outpatients, inpatients and operations in 1988].

Statistical studies were made on 1,766 outpatients, 657 inpatients and 687 operative procedures at our department in 1988. The most frequent diseases among the outpatients were urogenital infections followed by anomalies, tumors and stones. The major diseases among the inpatients were congenital urethral stenosis, hypospadias, vesicoureteral reflux, benign prostatic hypertrophy and bladder tumor. A total of 687 operations were performed. The five major operations were optic internal urethrotomy, hypospadias repair, transurethral resection of prostate, ureterocystoneostomy and transurethral resection of bladder tumor.

Ultrasonographically demonstrated nutcracker phenomenon: alternative to angiography.

We report on a child with nutcracker phenomenon, which is a possible cause of intermittent gross haematuria of unknown origin. Early serial ultrasound examinations can demonstrate the lesions. The merits and demerits of conventional angiographic imaging are also discussed.