Volumetric imaging: a potential tool to stage upper tract urothelial carcinoma.

PURPOSE: To investigate whether volumetric imaging of tumor vasculature can be used to phenotypically characterize advanced upper tract urothelial carcinoma, and if this technique can distinguish aggressive invasive tumors from non-aggressive superficial ones. METHODS: In a pilot study, two TaG1 and two T3G3 formalin-fixed paraffin-embedded (FFPE) tumor samples were examined using the DIPCO pipeline (Tanaka et al. in Nature Biomed Eng 1(10):796-806. https://doi.org/10.1038/s41551-017-0139-0 , 2017). Briefly, punch biopsies of FFPE tumors were deparaffinized, cleared, immunolabeled with the vessel marker CD34 and imaged with a light-sheet microscope. Thereafter, the three-dimensional (3D) vasculature of the tumors was analyzed and characterized using a specialized image processing software. RESULTS: We found that T3G3 tumors had increased CD34 density kurtosis and skewness compared to TaG1 tumors. This suggests that analysis of the 3D vasculature can distinguish between high-grade invasive and low-grade superficial tumors. CONCLUSIONS: Volumetric imaging of tumor samples may represent novel methodology that can complement conventional histopathology. Volumetric imaging enabled us to differentiate between invasive and non-invasive upper tract urothelial carcinoma. The method is of particular interest in diagnostic work-up of patients with upper tract urothelial carcinoma as previous findings indicate that volumetric imaging of vascular patterns could be used to differentiate superficial and invasive urothelial carcinoma, irrespective of if the tumor sample was deep or superficial. However, further and more extensive studies are required before this method can be applied clinically.

Angiogenesis in upper tract urothelial carcinoma associated with Balkan endemic nephropathy.

Upper tract urothelial carcinoma (UTUC) associated with Balkan endemic nephropathy (BEN) is characterized by a number of aberrations in cell-cycle regulation and apoptosis. The aim of this study was to detect angiogenesis-related marker(s) specific for BEN UTUC, and to examine the influence of HIF 1α upon angiogenesis and apoptosis in UTUC. Present investigation included 110 patients with UTUC, 50 from BEN region and 60 control tumors. Altered expression of VEGFR1 was more often present in control UTUC than in BEN tumors (p<0.005). It was associated with high grade, low and high stage, solid growth, and metaplastic change of control UTUC. Microvessel density assessed by CD31 (MVD CD31) was significantly higher in UTUC with lymphovascular invasion (p<0.05), and in BEN tumors with papillary growth (p<0.05). Discriminant analysis indicated that BEN and control tumors do not differ significantly in expression of angiogenesis related markers. The most important discriminant variable that determined control UTUC was expression of VEGFR1 (p=0.002). HIF 1α in UTUC significantly correlated with the low stage, papillary growth and expression of Bcl-2, Caspase-3 index, and MVD CD34 (p<0.001; 0.0005; 0.01; 0.005; 0.01, respectively). HIF-1α may be helpful marker in evaluation of UTUC, especially when combined with angiogenesis and apoptosis.

Value of preoperative superselective embolization of the isthmus in a patient with upper urinary tract urothelial carcinoma and horseshoe kidney.

Standard treatment for upper urinary tract urothelial carcinoma (UUTUC) implies the radical removal of all urothelium-lined tissue, which requires nephroureterectomy with bladder cuff removal. We report on a patient with a rare coincidence of UUTUC and horseshoe kidney in whom a preoperative angiography helped to identify and subsequently embolize an abberant isthmic feeding artery, which was located in between both collecting systems. Ischemic discoloration of the isthmus area facilitated resection and no major blood loss occurred. Preoperative superselective embolization of the isthmus as the renal split area can be an effective tool to facilitate nephroureterectomy in the case of a horseshoe kidney.

The impact factors on prognosis of patients with pT3 upper urinary tract transitional cell carcinoma.

PURPOSE: Stage 3 upper urinary tract transitional cell carcinoma is a heterogeneous disease including different tumor locations (pelvis vs ureter) and invasion patterns (renal parenchyma, peripelvic fat and periureteral fat). Unfortunately the outcomes of patients with pT3 disease with different invasion pattern are largely unknown. This study presents the clinical outcome of patients with pT3 disease with upper urinary tract transitional cell carcinoma. MATERIALS AND METHODS: We retrospectively reviewed the medical records of all patients with pT3 disease with upper urinary tract transitional cell carcinoma. Four patient groups were classified according to tumor location and tumor invasion pattern. Prognostic factors including age, gender, tumor grade, tumor size, tumor number, tumor location and microscopic finding of vascular invasion were analyzed with respect to disease recurrence and survival. RESULTS: A total of 72 patients were included in this study. The most common complaint and tumor relapse pattern were painless gross hematuria and distant metastasis, respectively. Patients with pT3 disease with superficial parenchymal invasion had better disease-free and recurrence-free survival than the other 3 groups. Initial tumor location (p = 0.02) and vascular invasion (p = 0.02) were independent factors for disease-free survival, and vascular invasion (p = 0.001) was the only predictive factor for recurrence-free survival. CONCLUSIONS: The present study demonstrated that patients with pT3 disease with superficial parenchymal invasion should be considered to have lower stage disease, and that vascular involvement is the only independent prognostic factor for patients with pT3 disease for disease-free and recurrence-free survival. Systemic adjuvant therapy should be recommended for patients with pT3 disease with vascular involvement.

Pathological function of prostaglandin E2 receptors in transitional cell carcinoma of the upper urinary tract.

The prostaglandin E2 receptor, EP4 receptor (EP4R), plays an important role in the development of transitional cell carcinoma of the upper urinary tract (TCC-UUT). However, the clinical significance of other EP receptors (EP1R-3R) is not clear. Furthermore, the pathological function of EP receptors in such patients is not understood. In the present study, we examined the expression of EP1R-3R in 101 TCC-UUT tissues by immunohistochemistry. Furthermore, we defined the relationship between cyclooxygenase (COX)-2 and EP receptor expression, proliferation index (PI), microvessel density (MVD), and expression of metalloproteinase-2 (MMP-2), urokinase-type plasminogen activator (uPA), and exon v6 containing CD44 isoform (CD44 v6) by multivariate analysis. The expression of EP1R, EP2R, and EP3R was positive in 20 (19.8%), 26 (25.7%), and 14 (13.9%) tumor samples, respectively. Expression of these receptors was not associated with pathological findings or survival. COX-2 and EP4R were independently associated with MVD and MMP-2, and uPA or PI and MMP-2, respectively. Other EP receptors were not influenced by any factors. Our results suggest that EP1R-3R play a minimal role in cancer progression in patients with TCC-UUT. On the other hand, EP4R regulates tumor progression via cancer cell proliferation and MMP-2, distinct from COX-2.

Angiogenesis in urothelial tumors of the upper urinary tract.

Angiogenesis is an essential process in the progression of malignant tumors. Tumors of the ureter and renal pelvis account for 5% of all urinary tract neoplasms. Little is known about angiogenesis in upper urinary tract urothelial tumors. We tried to demonstrate angiogenesis by using three endothelial markers CD31, CD34, von Willebrand factor and one pericytes marker (alpha-smooth muscle actin) in 26 cases. The pattern of CD31 immunolabelling was more complex and extensive than the vessel pattern shown by CD34 or factor VIII staining. In non-invasive tumors we observed that angiogenesis process is limited to connective tissue of tumor stroma. In the tumor area, the blood vessels stained with anti-CD31 had large lumen, thin walls and numerous branches, some of them being very thin. Pericyte covered vessels were branching of frequently into smaller, pericyte negative vessels.

[Prognostic implication of morphometric stromal parameters of renal pelvis and ureteral transitional cell carcinomas]. / Prognosticheskoe znachenie morfomekhanicheskikh parametrov stromy prekhodno-kletochnykh kartsinom lokhanki pochki i mochetochnika.

To evaluate prognostic value of morphometric studies of the stroma of transitional cell carcinomas of the renal pelvis and ureter, we studied retrospectively the data of primary examination and follow-up of 75 patients (49 males, 65% and 26 females, 35%; mean age 61.9 +/- 1.2 years) given radical surgical treatment for cancer of the renal pelvis and ureter. Five-year survival in the absence of tumor progression was 23%. Morphological examination diagnosed transitional cell carcinoma with invasion pT1, pT2, pT3 and pT4 in 3(4%), 15(20%), 47(63%) and 10(13%) cases and differentiation degree G1, G2, G3 in 31(41%), 15(20%) and 29(39%) cases, respectively. In addition to the standard morphological examination of the tumor, we made morphometry of stromal and tumor area, analysed composition and count of stromal effector cells (lymphocytes, eosinophilic and neutrophilic leukocytes, macrophages, mast and plasmic cells), the degree of stromal vascularization. Prognostic value of the above parameters was estimated according to significance of their correlation with postoperative survival of the patients. The survival correlated with the depth of cancer invasion (p = 0.005) and differentiation of tumor tissue (p = 0.006), high cell infiltration of tumor stroma is prognostically unfavourable (R2 = 0.03; F = 3.41; p = 0.069) as well as weak presentation of stromal component of the tumor (p = 0.056). The lowest survival was observed in patients with cancer of the renal pelvis and ureter with a great number of mast cells (p = 0.056), macrophages (p = 0.037) and neutrophils (p = 0.029) in the tumor stroma. According to the results of multiple regression analysis (R2 = 0.08; F = 5.42; p = 0.024), five-year postoperative survival most closely correlated with cancer invasion depth (p < 0.001), degree of tumor cells differentiation (p < 0.001) and number of macrophages infiltrating tumor stroma (p < 0.001). Significance of survival prognosis for patients with cancer of renal pelvis and ureter can be raised by estimation of mean number of free stromal cells and expression of stromal component.

Expression of metalloproteinase-2, metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 in transitional cell carcinoma of upper urinary tract: correlation with tumor stage and survival.

OBJECTIVES: To investigate the relationship between the expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 and pT stage or survival in patients with transitional cell carcinoma of the upper urinary tract. MMP-2 and MMP-9 are associated with tumor invasion in several malignancies. TIMPs exert an anti-invasive effect by blocking MMP activity. Recent studies have shown, however, that TIMPs can also stimulate cell proliferation and angiogenesis. METHODS: Tumor sections surgically removed from 91 patients were examined for expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 by immunohistochemistry. We also determined the proliferation index and microvessel density in each tumor and investigated the independent roles of these factors in tumor stage and survival using multivariate analysis. RESULTS: Of 91 tissue samples, 50, 51, 45, and 39 were positive for MMP-2, MMP-9, TIMP-1, and TIMP-2 expression, respectively. Tumors positive for MMP-2, MMP-9, and TIMP-1 exhibited a greater proliferation index than tumors with negative expression (P <0.001, P = 0.013, and P <0.001, respectively). The microvessel density of tumors positive for MMP-2 and TIMP-1 was greater than that of negative tumors (P <0.001). The expression of MMP-2, MMP-9, and TIMP-1 was an independent predictor of high pT stage. Cox proportional hazard analysis identified TIMP-1 expression as an independent factor for cause-specific survival (odds ratio 5.2, P = 0.011), similar to microvessel density, pT4, and lymph node metastasis. CONCLUSIONS: TIMP-1 expression correlated with pT stage and was an independent predictor of cause-specific survival. Our results suggest that TIMP-1 expression is a potentially useful tool for the selection of postoperative observation strategies in patients with transitional cell carcinoma of the upper urinary tract.

Evaluation of cell proliferation, apoptosis, and angiogenesis in transitional cell carcinoma of the renal pelvis and ureter.

OBJECTIVES: To investigate cell proliferation, apoptosis, and angiogenesis and their roles in transitional cell carcinoma (TCC) of the renal pelvis and ureter. METHODS: Formalin-fixed and paraffin-embedded tissue blocks from 42 patients with TCC of the renal pelvis and ureter were studied. Cell proliferation was assessed by Ki-67 immunostaining, and the proliferation index (PI) was expressed as a percentage of Ki-67-positive cells. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of TUNEL positive cells. Angiogenesis was evaluated by CD31 immunostaining, and microvessel density (MVD) was expressed as the average of the microvessel count. RESULTS: The PI ranged from 5.9% to 48.0% (median 20.03%), AI from 1.0% to 4.2% (median 2.26%), and MVD from 16.0 to 146.0 (median 56.88) in TCC of the renal pelvis and ureter. Statistical analysis revealed close associations of both PI and MVD with tumor stage and of AI with tumor grade. Our study demonstrated a strong relationship between PI and MVD, but did not show associations of AI with PI or MVD in TCC of the renal pelvis and ureter. CONCLUSIONS: It is suggested that the high activity of tumor cell proliferation with rich neovascularization may be related to the high malignant potential of the cancer, and evaluation of cell proliferation combined with angiogenesis may be useful in predicting the progression of the renal pelvic and ureteral TCC.

[Studies on vessel invasion by cancer of the renal pelvis and ureter].

The significance of vessel invasion by cancer of the renal pelvis and ureter was estimated with surgical specimens of 45 patients. The vessel invasion by cancer was observed in 25 out of 45 cases (55.6%). The incidence of vessel invasion increased with the grade of cancer and the extent of the primary tumor. The postoperative metastases by cancer was noted in 22 of the 25 patients with vessel invasion (88%) and in 4 of 20 (20%) patients without vessel invasion. The incidence of metastases in patients with vessel invasion was significantly higher than that without it (p < 0.01). The 5-year survival rate was 13.1% in the patients with vessel invasion and 80.6% in the patients without it (p < 0.005). Postoperative chemotherapy had no effect on the unfavorable outcome of the patients with vessel invasion. Therefore, vessel invasion by cancer may be one of the prognostic factors in renal pelvic and ureteral cancer. The patients with vessel invasion should be treated with more aggressive therapy to improve the poor prognosis.