Bladder transitional cell carcinoma: correlation of contrast enhancement on computed tomography with histological grade and tumour angiogenesis

Clinical Radiology (2005) 60, 215–223

Bladder transitional cell carcinoma: correlation of contrast enhancement on computed tomography with histological grade and tumour angiogenesis Q. Xiea, J. Zhangb, P.-H. Wud,*, X.-Q. Jianga, S.-L. Chena, Q.-L. Wange, J. Xub, G.-D. Chena, J.-H. Dengc Departments of aRadiology, bPathology, cUrology, The First Municipal People’s Hospital of Guangzhou, Guangzhou, dDiagnostic Imaging and Interventional Radiology, Tumour Hospital of Sun Yat-sen University, Guangzhou, and eFamily Plan Institution of Guangdong Province, Guangdong, People’s Republic of China Received 3 October 2003; received in revised form 29 April 2004; accepted 9 May 2004

KEYWORDS Bladder neoplasms; Tomography; Spiral computed; Neovascularization/ patholgic; Microvessel density (MVD); Vascular endothelial growth factor (VEGF)

AIM: To investigate the correlation between the degree of contrast enhancement of bladder cancer in the early enhanced phase of helical computed tomography (CT) and microvessel density (MVD), vascular endothelial growth factor (VEGF) and histological grade. MATERIALS AND METHODS: Sixty-five patients with transitional cell carcinoma of the bladder were examined by incremental unenhanced CT and helical CT at 40– 45 s after initiation of intravenous administration of contrast medium before surgery. The CT density in Hounsfield units of bladder carcinomas were measured in the middle of the maximum diameter section of the cancer lesions on unenhanced and enhanced CT. The degree of contrast enhancement of the tumour was determined as the absolute increase in Hounsfield units. Histological grade, VEGF and MVD were analysed for each cancer. The Pearson and Spearman correlation tests were used to determine the strength of the relationships between CT enhancement and histological grade, VEGF expression and MVD. RESULTS: Different degrees of enhancement were observed in 91 cancers during the early enhanced phase of helical CT. Mean MVDs and mean CT enhancing values of different histological grade groups were statistically different ðp , 0:001Þ: A positive correlation was found in the CT-enhancing value of bladder cancer and MVD (Pearson correlation test; r ¼ 0:938; p , 0:001) and histological grade (Spearman rank correlation; r ¼ 0:734; p , 0:001). VEGF of bladder cancer did not correlate with the change in CT attenuation (Spearman rank correlation; r ¼ 0:087; p ¼ 0:410) and MVD (Spearman rank correlation, r ¼ 0:103; p ¼ 0:330). CONCLUSION: In bladder cancer, the degree of contrast enhancement during the early enhanced helical CT is correlated with the MVD and histological grade of tumour. It is possible that MVD is the histopathological basis of early contrast enhancement of bladder cancer. q 2005 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Introduction *Guarantor and correspondent: P.H. Wu, Department of Diagnostic Imaging and Interventional Radiology, Tumour Hospital of Sun Yat-sen University, Dong Feng Dong Road, Guangzhou 510060, People’s Republic of China. Tel.: þ86-2087343271; fax: þ 86-20-87343270. E-mail address: [email protected]

Bladder cancer is the most common cancer of the urinary tract, and transitional cell carcinoma (TCC) accounts for . 90% of bladder cancers.1 TCC is classified histopathologically into three types: superficial diseases (papillary tumours), superficially invasive tumours (PT1, PTa tumours), and

0009-9260/$ - see front matter q 2005 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.crad.2004.05.009

216 deep muscle invasive tumours (stages T2 – T4).2,3 The existence of different forms of bladder cancer was originally thought to reflect sequential development from superficial diseases to deeply invasive carcinoma.2 However, superficial bladder cancers represent a heterogeneous group of tumours, and about 60% of them will recur after transurethral resection.4 Some of them will progress to invasive and/or metastatic tumours and are therefore potentially lethal.3 The prognosis in a given patient is difficult as there is no single factor that can be used consistently for prognostic information. Some previous studies suggest that tumour angiogenesis was an independent prognostic factor of bladder cancer.5 – 8 Microvessel density (MVD), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression by human TCC have also been investigated as prognostic markers.7,9 – 14 Angiogenesis, the process by which tumours induce a blood supply, is crucial for growth and progression.7,15 Taking this into account, potential anti-angiogenic therapies may have an important role in the treatment. Folkman noted that a therapeutic approach directed against tumour angiogenesis would not result in cure, but would likely prevent tumour expansion and induce a period of dormancy rather than sustained regression.16 The study of tumour angiogenesis and response of tumours to anti-angiogenic therapies is of intense clinical and research interest,17 – 28 and there are major implications for imaging with ultrasound, computed tomography (CT), magnetic resonance imaging (MRI) and other methods.17 – 25 In a previous study of bladder cancer staging with helical CT, we found that different degrees of enhancement were observed in TCC in early enhanced phase of CT, and the CT attenuation was related to histological grade.29 In order to investigate histopathological basis of cancer enhancement in early contrast CT, we developed a pelvic imaging protocol and performed helical CT for patients with bladder TCC.

Materials and methods Patients One hundred and sixty-seven patients were examined prospectively from March 1996 to March 2003. All patients had a diagnosis of bladder TCC and underwent pelvic helical CT before treatment. Sixty-five of these patients fulfilled the entry criteria29 of the study, namely: (1) patients under-

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went partial cystectomy (27 with superficially invasive tumours, T1) and radical cystectomy and pelvic lymph node dissection (38 with greater muscle invasive tumours, T2 – T4) within 1 week after CT examination; and (2) the cystectomy specimens and dissection lymph nodes were available and labelled for histopathological study. The patient population included 47 men and 18 women with an age range of 34 –86 years old (mean age 67.6 years). The study was approved by the Research and Education Information Department of the First Municipal People’s Hospital of Guangzhou for clinical research and medical ethics. Informed patient consent was not required.

CT Protocol Helical CT was performed with Picker Helicat II machine. All patients were given 500 ml of 3% oral iodinated contrast material (Angiography, Schering, Guangzhou company, China) for bowel opacification beginning 60 min before CT examination. Over a period of 30 min before imaging, patients ingested at least 500 ml water to allow optimal urine filling of the bladder. Patients were examined in the prone position. After an incremental unenhanced study of pelvis was obtained, enhanced helical CT was started from the bottom of pelvis to the top of bladder. A total of 80 – 100 ml nonionic contrast medium (Ultravist 300, Schering, Guangzhou company, China) was administrated intravenously with an automatic injector at a flow rate of 2 ml/s. In a pilot study, bolus tracking at the maximum diameter axial section of tumour was performed in five patients (age range: 46 –78 years, four men, one woman). Beginning with contrast agent injection, 8 – 12 images were acquired every 5 – 8 s with 1 s acquisition time. The enhancement value (in Hounsfield units) of tumour was recorded. The peak tumour enhancement occurred at 42 – 54 s after initiation of intravenous administration of contrast medium. Thus, in the study group, early enhanced helical CT was started at 40 – 45 s after initiation of injection of contrast medium by using 220 mA, 120 kVp, and 5- or 10-mm collimation with a pitch of 1.5. The CT attenuation in the middle of the maximum diameter section of the cancer was obtained before and after contrast enhancement with the same size of regions of interest (ROI) for the same tumour. The ROI (1 – 20 mm2) were drawn independently by two radiologists (Q.X. and G.D.C. separately), differences in measurement were resolved by consensus. One measurement was chosen for each tumour before and after contrast

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enhancement. The absolute increase in Hounsfield units was recorded as the CT enhancement value.

was made between the CT enhancement value and histological grade, VEGF expression and MVD.

Histopathological study

Statistical analysis

Tissue blocks in the middle of the maximum diameter section of tumours were sectioned. Haematoxylin and eosin (HE) stained slides were assessed on standard histomorphometric techniques, on paraffin-embedded 4 mm thick sections. Immunohistochemical reactivity was tested for VEGF monoclonal antibody (Dako, Glostrup, Denmark) and endothelial cell labelling (CD34) monoclonal antibody (Dako), using the indirect streptavidin/peroxidase labelling techniques. Dark brown staining in cytoplasm or membrane was judged as positive reaction. VEGF positive reaction was evaluated and graded by light microscopy (Olympus, BH-2, Tokyo, Japan) as follows: þ ¼ positive reaction less than 5% of the inner area of the lesion,þ þ ¼ positive reaction in 5 – 14% of the inner area of the lesion,þ þ þ ¼ positive reaction in 15 – 50% of the inner area of the lesion,þ þ þ þ ¼ positive reaction more than 50% of the inner area of the lesion. MVD quantification was the technique described by Weidner et al.30 with a few modifications31,32 with light microscopy (Olympus, BH-2, Tokyo, Japan) on CD34 stained slides. Clusters of stained endothelial cells distinct from adjacent microvessel, tumour cells, or other connective cells were counted as one microvessel. Any vessel lumen larger than eight red cells or containing medial membrane and elastic fibres in the wall was not counted. The area of highest MVD of the tumour was found by scanning at £ 40 or £ 100 magnification, and then the single field with the highest number of microvessels at £ 200 magnification was identified. The MVD was expressed as the average count of the five areas randomly at £ 200 magnification field.

Mean MVD and mean CT enhancement value of different histological grade groups were assessed by analysis of variance (ANOVA). Chi-square test was used to compare VEGF expressions with histological grade. Correlations between the parameters (i.e. CT enhancement value, histological grade, and VEGF expression) were evaluated with Spearman rank correlation test. The relationship between CT enhancement value and MVD was investigated using Pearson correlation test. A p-value of less than 0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed with SPSS 11.0 software package (SPSS Inc.).

Radiological– pathological correlation All cystectomy specimens were examined by two pathologists (J.Z., J.X.). The information about the number, location and size of cancer was recorded. All HE stained slides and immunohistochemical stained slides were analysed by a single pathologist (J.Z.) who had no knowledge of patient’s data. Histological grading was performed according to the scheme proposed by the World Health Organization on HE stained slides of formalin-fixed and paraffin-embedded tumour issue. CT films were interpreted independently by one radiologist (Q.X.) who had no knowledge of the final pathologic results. In each cancer, one-to-one correlation

Results Ninety-one cancers were found and resected in surgery. The histological grades of the 91 resected tumours were I/III in 21, I –II/III in 10, II/III in 24, II – III/III in 21, III/III in 15. On enhanced helical CT images obtained at 40 – 45 s after initiation of contrast medium injection, 91 tumour lesions were detected. They demonstrated homogeneous enhancement without necrosis and liquefaction (Figs. 1 – 3). CT enhancement values were 5 – 70 HU. Bladder wall demonstrated no or slight enhancement (CT enhancement value was 0 – 5 HU), and there was no contrast medium in the urine of bladder. Table 1 lists the average CT enhancement values of tumours of different histological grade carcinomas. CT enhancement values increased from histological grade I to III (Figs. 1 –4), and the difference of average enhancement values was statistically significant (ANOVA, F ¼ 27:081; p , 0:001; Table 1). MVDs of 91 bladder cancers were 20 –136. Table 1 lists the average MVDs of cancers of different histological grades. Average MVDs increased from histological grade I to III (Figs. 1 – 3), and the difference of average MVDs was statistically significant (ANOVA, F ¼ 27:789; p , 0:001; Table 1). Table 2 lists the VEGF expression of the bladder cancers. The difference of VEGF expression was not statistically significant from histological grade I to III (Chi-square test, x 2 ¼ 22.427, p ¼ 0:130). MVD of bladder cancer did not correlate with VEGF expression (Spearman correlation, r ¼ 0:103; p ¼ 0:330). The CT enhancement value of bladder cancer

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Figure 1 Grade I bladder transitional cell carcinoma. a. Tumour shows a soft mass on the right wall of bladder on plain CT (arrow). b. 40 s after administration of contrast agent, pelvic SCT scans show slight enhancement of the tumour (CT enhancing value ¼ 8 HU). c. CD34 stained specimen demonstrates poor distribution of microvessel (dark brown, mag. £ 100), average MVD ¼ 20 (mag. £ 200).

and MVD showed a close positive correlation (Fig. 5, Pearson correlation, r ¼ 0:938; p , 0:001). CT enhancement value of bladder cancer correlated with histological grade (Fig. 4, Spearman rank correlation, r ¼ 0:734; p , 0:001). The CT enhancement value of bladder cancer did not correlate with VEGF expression (Spearman rank correlation, r ¼ 0:087; p ¼ 0:410).

Discussion Malignant solid tumour growth and metastasis depend upon the development of a neovasculature in and around the tumour.33 – 35 This process, called angiogenesis, is the formation of new microvessels from the existing vascular network.34 It has been reported that tumour angiogenesis is a prognostic

Table 1 Histologcal grade, CT enhancement value and MVD (X ^ S) Histological grade

No. of lesions

CT enhancing value

MVD

I I–II II II–III III

21 10 24 21 15

13.9 ^ 8.1 19.7 ^ 8.1 30.3 ^ 13.9 30.6 ^ 10.9 51.7 ^ 12.2

28.4 ^ 6.8 37.2 ^ 10.5 46.7 ^ 15.4 47.8 ^ 15.2 90.3 ^ 33.7

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Figure 2 Grade II bladder transitional cell carcinoma. a. Tumour shows a soft mass on the left-front wall of bladder on plain CT. b. 40 s after administration of contrast agent, pelvic CT scan shows moderate enhancement of tumour (CT enhancing value ¼ 22 HU). c. CD34 stained specimen demonstrates moderate dense distribution of microvessel (dark brown, mag. £ 100), average MVD ¼ 38 (mag. £ 200).

factor determining the biological behaviour.36 – 38 Bladder cancer, like all solid malignancies, also relies on the angiogenesis to grow progressively and metastasize efficiently.9,32,39 – 42 MVD has been Table 2 VEGF expression in different histological grades Histological grade

VEGF expressions 2

þ

I I–II II II– III III

2

5 2 2 1 2

Total

2

12

Total

þþ

þ þþ

þ þ þþ

5 4 11 11 7

6 2 10 6 2

5 2 1 3 2

21 10 24 21 15

38

26

13

91

extensively evaluated as a prognostic factor for TCC and correlated with lymph node metastasis, disease recurrence and survival.9 – 11,32 VEGF and bFGF expression by human TCC has also been evaluated.5,8,12 – 14,43 Elevated expression of VEGF in human tumour biopsies, as well as the rise of VEGF levels in urine or serum, have been reported to be independent prognostic and predictive factors of recurrence and disease progression in patients with urothelial cancer.8,12 – 14,43 Over-expression of bFGF mRNA in radical cystectomy specimens also independently predicted poor outcome for patients who received neoadjuvant chemotherapy.5 Without new blood vessel proliferation, tumour growth is stunted. This concept has led to the successful development of a variety of experimental therapies

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Figure 3 Grade III bladder transitional cell carcinoma. a. Plain CT shows tumour (arrow) on the back wall of bladder and a large perivesical lymph node (arrowhead). b. 45 s after administration of contrast agent, pelvic CT scan shows strong enhancement of tumour (CT enhancing value ¼ 63 HU) and large lymph node. Pathologic slides showed evidence of lymph node metastasis. c. CD34 stained specimen demonstrates high dense distribution of microvessel (dark brown, mag. £ 100), average MVD ¼ 93 (mag. £ 200).

designed to inhibit the growth of tumour neovascularity.15 – 17,26 – 28 Neovascularization within tumours can be diagnosed using histological techniques to determine MVD, VEGF expression and bFGF.5,7 The technique to assess MVD has been regarded as the gold standard in the assessment of tumour angiogenesis. But this requires removal of tumour. The process is complex and takes a long time. The results only reflect the angiogenesis of a small area of tumour, and is limited to use for serial monitoring of tumour response in human patients. Histological techniques can be used to assess angiogenesis only on condition that pathology specimens are obtained. Because of these limitations, some investigators have used imaging methods, which are reproduci-

ble, non-invasive, rapid and quantitative to assess neovascularization of cancer, and have compared imaging findings with the gold standard MVD. Studies of uterine, breast, lung and liver cancer have shown that contrast-enhanced dynamic MRI or CT had the potential to evaluate neovascularization of tumour in patients without the need to obtain biopsy specimens.20 – 25 Contrast enhancement of any organ or lesion depends on the size of the vascular and interstitial compartments because intravenous water-soluble contrast agents are distributed between these two spaces.25 After intravenous administration during CT, contrast agents are rapidly distributed by diffusion between the vascular and the interstitial spaces.25 The degree of contrast enhancement of a

Bladder transitional cell carcinoma

Figure 4 CT enhancement values (absolute increase in Hounsfield units) is plotted against histological grade for bladder cancers.

lesion depends on the vascular density of the lesion, as well as on the permeability of the microvasculature.25,44 Early enhancement of water-soluble contrast medium is believed to be caused by the density and distribution of tumour microvessel.22 Enhancement patterns of bladder cancers by CT are firmly established after the intravenous administration of water-soluble contrast agents. Bladder cancers receive blood only from the artery, the attenuation of bladder cancer on the early contrast enhanced CT at 40 – 45 s after initiation of contrast agent injection may represent vascularity (i.e. MVD). The present results indicate that the CTenhancing value of bladder cancer is positively correlated with MVD (r ¼ 0:938; p , 0:001) and

Figure 5 CT enhancement values (absolute increase in Hounsfield units) is plotted against MVD for bladder cancers.

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histological grade (r ¼ 0:734; p , 0:001). These results suggest that images acquired at 40 – 45 s after initiation of contrast agent injection give similar information to histological assessment of tumours. Although these images were insufficient to visualize tumour microvessels directly, because their diameter is approximately 2 – 5 mm, CT enhancement of bladder cancer had the potential to assess neovascularization, in which an increase in the number of microvessels within a given tumour tissue volume was likely to lead to increased tissue enhancement. Histopathological study showed that MVDs of bladder cancer correlated with its histological grade, which is in agreement with the earlier literature.42 This result may suggest a correlation between higher grade or more aggressive bladder cancer and a higher MVD. So in the early enhanced phase, the increased CT enhancement value of cancer lesion corresponds to the increased histological grade. However, the present results also showed that the VEGF expression did not completely correlate with the MVD. In some cancers, VEGF expression appeared negative or poorly positive with high MVD cancer lesions, and strongly positive with low MVD. There may be many reasons for this discordance, angiogenesis is not determined by a single factor but is regulated by the balance between stimulatory and inhibitory factors released by the tumour and its microenvironment.13,45 Superficial and invasive bladder cancer use different angiogenesis pathways during development.46,47 For example, expression of VEGF is four-fold higher in superficial tumours than in invasive tumours.47 Inflammatory cells are also a source of VEGF.12 Therefore, the effects of other angiogenic factors, angiogenesis inhibitors and inflammatory pathology should be evaluated, and the effects of receptors for VEGF should also be evaluated. Moreover, the difference in time interval between the CT study and the surgery may be a cause of the discordance. There are potential limitations of this study. First, only patients who underwent partial or complete cystectomy within 1 week of CT were included this study. Therefore, the sample population was not randomly selected. Second, one ROI was used instead of multiple ROIs to calculate the CT attenuation of each cancer by two radiologists (Q.X., G.D.C.) separately according to their experiences. ROI analysis is prone to errors from operatordependent choice of size and position. Third, during histopathological assessment, analysis is usually limited to one or a few sections. It is presumed that MVD in this section represents the MVD estimated by CT enhancement values on the CT image. This analysis has a potential sampling error. Finally, the

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arterial input function, which is influenced by the injection rate and the cardiac output and which may affect the increase in CT attenuation, was not accounted for. To keep the study simple and to reduce errors, tissue blocks for histopathological analysis were obtained from the middle of the maximum diameter section of tumours. The ROI for CT attenuation was also obtained from the maximum diameter of the axial sections of the tumours. Differences in the measurement of CT attenuation were resolved by consensus. In summary, the degree of enhancement of bladder cancer was found to be correlated positively with MVD and histological grade on early enhanced CT. It is possible that MVD is the histopathological basis of enhancement of bladder cancer at early contrast enhanced CT. Early enhanced CT may serve as a non-invasive method to evaluate tumour angiogenesis. In this regard, contrast-enhanced CT may play a useful role in the selection and follow-up of patients who receive anti-angiogenesis therapy.

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