Characterization of histological subtypes of clear cell renal cell carcinoma using contrast-enhanced ultrasound (CEUS)

Abstract

Introduction: The aim of this study was to analyze the histological subtypes of clear cell renal cell carcinoma (RCC) examined by means of contrast-enhanced ultrasound (CEUS) and a second generation blood pool agent (SonoVue^®, Bracco, Milan, Italy) during the pre-operative phase.

Materials and Methods: 29 patients with histologically proven subtypes of clear cell RCC were examined. A total of three patients were diagnosed with highly differentiated clear cell RCC, 21 out of 29 cases with moderately differentiated clear cell RCC and five out of 29 patients had insufficiently differentiated clear cell RCC. An experienced radiologist examined the patients with CEUS. The following parameters were analyzed: maximum signal intensity (PEAK), time elapsed until PEAK is reached (MTT), local blood flow (RBF), area under the time intensity curve (AUC) and the signal intensity (SI) during the course of time. For the groups all comparisons are made based on healthy renal parenchyma.

Results: In the clear cell RCC significant differences (significance level p < 0.05) between cancerous tissue and the healthy renal parenchyma were noticed in all four parameters. Therefore, the clear cell RCC stands out due to its reduced blood volume. However, it reached the PEAK reading relatively rapidly and its signal intensity was always lower than that of the healthy renal parenchyma. In the arterial phase retarded absorption of the contrast agent was observed, followed by fast washing out of the contrast agent bubbles.

In all three histological subgroups no significant differences were noticed in PEAK and SI. However, the diagrams showed the possible bias, that the group of the insufficiently differentiated clear cell RCC had the highest PEAK-value and the highest signal intensity when compared with highly and moderately differentiated clear cell RCC.

Conclusion: Our study suggests that CEUS may be an additional tool for non-invasive characterisation and differentiation of the three histological subtypes of clear cell RCC. Furthermore, it seems to have an additional diagnostic value in daily clinical.

1 Introduction

The clear cell renal cell carcinoma (RCC) has an incidence of 3% of all malignant neoplasms [7, 27]. Therefore, it is the third most common tumor of the urinary tract in Europe, followed by prostate and urinary bladder carcinoma. Europe is estimated to have the highest incidence worldwide [6 , 43]. With 85% of all renal carcinoma in adults, it is the most common malignant renal carcinoma. It occurs most commonly in patients between 60 and 70 years of age with a ratio of 3:2 [4 , 41],gender distribution shows a higher incidence in male than in female. More than 40% of patients decease within the first 5 years after diagnosis [19, 30]. Therefore, the clear cell RCC is the sixth most common of all cancer-related causes of death, and also the deadliest of all tumors that originate from the urinary tract [18]. Aggressive growth and thus infiltration of the peri-renal capsule or renal sinus occurs in approximately 20% of cases preventing metastatic spread in the abdomen through gerota’s fascia. Occasionally tumors are found in the renal vein, which may infiltrate the inferior vena cava or even the right atrium.

Following histopathological criteria, the RCC is subdivided into various groups in which the clear cell or conventional RCC occurs in 70 to 80% of malignant neoplasms, thus representing the most common tumor type in this group. It is mostly solitary, multifocal in one kidney in 4% and bilateral in 0.5% to 3%. The clear cell RCC can reach sizes of up to 15 cm, and has a worse prognosis than the papillary or chromophobe RCC. As a consequence, the exact clinical diagnosis is of paramount importance for the patient’s outcome.

The current 2014 European guidelines suggest contrast-enhanced computer tomography and magnetic resonance imaging as the preferred methods of medical imaging of clear cell RCC [32]. In special clinical cases CEUS is recommended [8 , 37]. Unlike contrast-enhanced computer tomography and magnetic resonance imaging contrast agents, the ultrasound contrast agents remain intravascular. According to the literature, in less than 0.002% of cases CEUS caused anaphylactic reactions [34, 38]. CEUS is a fast, low-risk and cost effective way for diagnosis of RCC [25].

2 Material and methods

Between June 2006 and June 2009 a total of 29 patients diagnosed with proven subtypes of clear cell RCC were examined using contrast-enhanced ultrasound. In advance, the local ethics committee approved this study. All study data were obtained according to the principles of the Helsinki/Edinburgh Declaration of 2002. Three out of 29 cases were highly, 21 moderately and five mildly differentiated RCC. After given informed consent, all patients were examined by means of CEUS by one single radiologist with more than 10 years of professional experience.

The sonographic examination was carried out with a Siemens (Germany) Acuson Sequoia 512 ultra sound device and a sonic head 4C1 [12 , 37]. The examinations were carried out by means of the Cadence^TM contrast pulse sequencing (CPS) technology developed to detect non-linear response of micro bubbles.

The contrast agent SonoVue^® (Bracco; Milan, Italy) was administered via a peripheral needle larger than 20–22 G, and was rinsed subsequently with 5 to 10 ml of 0.9% saline. The amount of contrast agent was between 1.6 and 2.4 ml in most cases. The quantity was doubled up to 4.8 ml or reduced to 1.0 ml [20]. After administration of the contrast agent craniocaudal, transverse and longitudinal images as well as videos lasting approximately 2 to 3 minutes were taken. A distinction of two phases – the arterial and the venous phase – was made in the process. The arterial phase covered the first 10 to 30 seconds, the venous phase the first 30 to 45 seconds after the first microbubbles were flooded in the area to be examined. The gathered data were mathematically processed by Qontrast Esaote Sp (EC mark no 0051, class IIA), a program developed for this particular purpose. In order to keep accidental errors as low as possible, three tests in the carcinoma of each patient were carried out during the following periods:

When the first bubbles of the contrast agent flooded the tumor

30 seconds after administration of contrast agent

60 seconds after administration of contrast agent

120 seconds after administration of contrast agent

Only the first three time intervals were considered relevant for statistical analysis. Allocating the images to the particular time when they were taken was a further aspect. As sonography is a real time examination and the quality of the images depends – as in this case - on the cooperation of the patient. The tissue to be examined was not always clearly visible at exactly the respective point in time. For this reason, a divergence with respect to the timeline of 12 seconds could not be avoided.

The parameters examined for each time interval were as follows:

PEAK reading: maximum signal intensity

Time to peak (TTP): time elapsed until maximum signal intensity is reached

Regional blood volume (RBV)

Regional blood flow (RBF)

Mean transit time (MTT)

Area under the curve (A.U.C.): area under the intensity curve

SI: Signal intensity over time.

Herein, RBV is proportional to the area under the time intensity curve (A.U.C.). RBF represents the ration between RBV and MTT.

The parameters were automatically calculated according to a formula based on the previously chosen Gamma-Variate Mode: SI (t) = PEAK*(t/TTP) (ß*TTP)e-ß*(t-TTP). Timing for the second measurement (TTP 2) was calculated based on both measurement times and the assumption, that the PEAK reading will be reached after 30 seconds (TTP 1), whereby the parameter ß of the model could be approximated by means of the Newton method.

Three measurements were taken in every patient both in tumor as well as in healthy renal parenchyma at each time interval. A mean value was calculated for each individual patient. This value was entered into the statistic model. It was based exclusively on the mean value of the corresponding time period. In order to achieve this, four out of the five above stated parameters were used, PEAK, A.U.C., MTT and RBF. These parameters provide essential information on time of flooding, retention period and washing out of contrast agent. Mean values were calculated for tumor and healthy tissue at each time interval. A Kruskal-Wallis test was used because of the small numbers of patients in each sub-group. The statistical analysis was carried out by R (version 3.0.1) RCoreTeam 2013. Bilaterally testing was done and a significance level of 5% was determined.

3 Results

Three male patients with an average age of 77.3 years were diagnosed with highly differentiated clear cell renal cell carcinoma. The tumors had an average size of 3.67 cm. As the B-Mode ultrasound shows, the malignant tumors were inhomogeneous and equally echoic with hyperechoic components as they increased in size. One of these three cases had a central necrosis.

21 patients diagnosed with moderately differentiated clear cell RCC constituted the largest group with 13 men with an average age of 65.6 years and 8 women with an average age of 65.5 years. Tumor size averaged 3.8 cm in the first and 4.3 cm in the second group, repsectively. In sonography, 12 tumors were hyperechoic, seven showed the same reaction to echo and two were hypoechoic. A central necrosis existed in 5 out of 21 cases.

Five patients were diagnosed with insufficiently differentiated clear cell RCC. Among them two were male with an average age of 63.5 years, and three female with an average age of 54.7 years. Tumor size averaged 9.5 cm in the first, and 11.5 cm in the second group, respectively. In B-Mode ultrasound tumors were inhomogeneous. Three were equal in their reaction to echo, one was hyperechoic and another one hypoechoic. In 4 out of 5 tumors a central necrosis could be seen in ultrasonography (Tables 1 and 2).

Data gathered by means of CEUS showed significant differences in all four parameters of the whole group of clear cell RCC. A level of p < 0.05 was considered significant for all statistic findings. A p-value of 0.00 was calculated for the PAEK, A.U.C. and RBF and 0.02 for MTT (Figs. 1–4). According to these findings, the clear cell RCC stands out because of reduced blood volume and reached the PEAK value relatively fast. As shown in Fig. 5, delayed absorption of contrast agent characterized tumor tissue with microbubbles being removed more rapidly afterwards. Therefore, the clear cell RCC showed weaker signal intensity than the surrounding healthy renal parenchyma over the whole period.

Statistical data with respect to PEAK of the individual subtypes were determined subsequently (Fig. 6). Due to the small number a non-parametric Kruskal-Wallis variance analysis was carried out, which did not reveal a significant difference between the individual histologic subtypes with a p-value of 0.27 and a significance level of p < 0.05. Assuming that the light grey line reflected the median or the continuous reaction of healthy tissue and therefore did not show any difference between tumor and healthy renal parenchyma. It can be concluded that individual subtypes did not have a lower PEAK readings (statistics: median) compared with the starting point 0. The insufficiently differentiated clear cell RCC is the one histological group, which displayed a reaction similar to that of healthy tissue. However, it was more hypoechoic than the surrounding renal parenchyma. The gamma variation curves for each patient were estimated based on PEAK readings when first bubbles appeared, the “30th second” as well as the individual moment when measurements were made. It was assumed that PEAK reading would be reached after 30 seconds. Based on individual measurement times and this assumption, the moment of the second measurement could be calculated and in the process the missing parameter could be approximated by means of the Newton method. The various moments after reaching the PEAK reading were observed in Fig. 7: 15, 30 and 60 seconds. The signal intensity during these moments was calculated by means of the adjusted model for each patient.

The test showed the following p-readings for the subgroups at the respective moments with respect to the histological subtypes of clear cell RCC. The findings did not show a significant difference between the histological subtypes of the clear cell RCC with respect to the PEAK readings and the signal intensity (Table 3).

4 Discussion

The correlation between the dignity of a renal tumour and its reaction to CEUS became an item of scientific interest as early as the 1990’s. Chiba stated that small renal tumors may be successfully detected and diagnosed by means of CEUS [5]. Ascenti et al. came to the same conclusion [2]. This aspect was also endpoint of this study.

In the current study, the most frequent malignant renal tumors were quantitatively examined and compared based on parameters gathered through CEUS and SonoVue^®. Clear distinctions with respect to PEAK, A.U.C., MTT and RBF in the group of clear cell RCC could be made. Compared with healthy renal parenchyma, clear cell RCC showed a reduced blood volume. One possible explanation for this finding could be the frequently existing haemorrhages and necroses that was found in 10 out of 29 patients. At the same time, there was a minimal difference in PEAK values, suggesting an increased metabolism of tumor cells. As demonstrated in Fig. 5, clear cell RCC featured delayed absorption of contrast agents during the early phase and faster washing out of microbubbles during the later phase. The delayed absorption of contrast agents may be explained by a lower rate of small caliber blood vessels. The faster washing out of microbubbles would be possible either in arteriovenous fistulas or in normal physiological reflux in the renal vein [11 , 40]. Throughout this time period, this tumor type showed lower signal intensity than healthy renal parenchyma. Clear cell RCC presented as heterogeneous and hypoechoic compared with the surrounding tissue. Additional quantitative differentiation between the histological subtypes of clear cell RCC (Figs. 6 and 7, Table 3) did not reveal significant findings. Nevertheless, the figures indicated that the insignificantly differentiated clear cell RCC tended to have the highest signal intensity as compared to the highly and moderately differentiated clear cell RCC. The insufficiently differentiated clear cell RCC therefore most resembled healthy renal parenchyma, allowing conclusions concerning elevated metabolism and aggressive growth of this tumor type.

A possibly positive correlation between dignity and characteristics of renal tumors in contrast-enhanced sonography was examined in other studies.

Fan et al. examined malignant and benign renal tumors with a diameter of no more than 5 cm with CEUS [16]. The authors concluded that the inhomogeneous and hyperechoic image during the late phase was highly significant for diagnosing RCC (specificity 100%, sensitivity 59.1%).

Dong et al. assessed, how differently clear cell RCC reacted to contrast agents [15]. The authors described four different patterns of contrast agent absorption and washing out, and compared these with the histological properties of the tumors.

However, in the present study a different behavior pattern of the clear cell RCC was observed: i.e. slow washing and fast washing out, the intensity curve of which demonstrated the same parallel progression as healthy renal parenchyma (Fig. 5).

Shen et al. reached similar conclusions [36]. Different time intervals for flooding of contrast agent (1), washing out of contrast agent (2), and late phase (3) were determined in the study conducted by Dong et al. (1).

Xu et al. differentiated in their study between renal malignancies with the help of CEUS [44]. The authors observed that contrast agent was absorbed differently, i. e. in increased or equal quantities by the majority of the clear cell RCC during the early phase and when the contrast agent was removed during the late phase.

Ignee et al. also examined renal neoplasms with CEUS [28]. The majority of the RCC showed increased signal intensity during the early phase (up to 30 seconds). In a few cases only a reduced absorption of the contrast agent was observed. In addition, the reaction to the contrast agent in the arterial phase was different from that in the late phase (1 to 3 minutes), in which two thirds of the lesions showed reduced signal intensity. Furthermore, the authors reported hypervascularity in the majority of clear cell RCC and hypovascularity in a third of papillary RCC, both of which influenced washing out of contrast agent bubbles to a high degree.

In contrast to the present study, findings by Aoki et al. suggest a higher perfusion of solid and cystic renal tumors [1, 9].

Zhou et al. examined the reaction of clear cell RCC and angiomyolipoma with CEUS [45]. Contrary to the present study, an increase in signal intensity was observed in all malignancies. The afore-mentioned studies suggested a positive correlation between histological subtypes and reaction to contrast agents displayed by RCC.

In the study performed by Cai et al. various malignant and benign renal carcinoma were examined by means of SonoVue^® and compared according to quantitative assessment of enhancement of the tumors [3]. Following parameters were assessed: arrival time (AT), TTP, wash-out time (WT), highest signal intensity (IMAX) and signal intensity at 60 seconds (I₆₀). The authors concluded that the malignant renal neoplasm group had lower readings for AT and TTP but higher readings for WT, IMAX and I₆₀ compared with benign renal carcinoma. Contrary to the present study, renal carcinoma were grouped together into two main groups without conducting further quantitative parametric analysis with respect to the histological subtypes of malignant and benign neoplasms.

Oh et al. examined and compared RCC (33 of which were clear cell RCC and 5 papillary RCC) and 11 angiomyolipomas by means of SonoVue^® [33]. In contrast to the present study, the maximum diameter in both groups was determined at 4.0 cm. The majority of the RCC featured both heterogenic absorption and wash out of the contrast agent during examination. Just on half of malignant neoplasms featured accumulation of contrast agent in the periphery of the lesion. Further differentiation between histological subtypes of clear cell and papillary RCC was not made.

The afore-mentioned studies indicate a positive correlation between histological subtypes and reaction to the contrast agents of the RCC.

Gerst et al. examined patients with malignant and benign renal carcinoma with the help of CEUS. The authors concluded that both speed with which the contrast agent is absorbed and vascular supply provided no information on histologic types. Furthermore, it was argued that the arterial phase alone and TTP were inconclusive. However, in this study the small number of patients involved was a limiting factor [17].

Haendl et al. investigated the reaction of RCC, oncocytoma and urothelial cell carcinoma with CEUS [21]). The author concluded that RCC did not display typical perfusion behavior in CEUS. However, the average variable tumor size of 6.54 cm measured with sonography was not addressed as a possibly disturbing factor, a well-known fact that may significantly influence the reaction of RCC to contrast agents [29, 31].

Setola et al. argued to the contrary and saw no benefit of CEUS in the evaluation of large renal tumors [35]. According to the authors, there was a benefit when smaller renal lesions were differentiated. As mentioned before, tumor size is an essential factor of quantity and quality in CEUS.

As the above mentioned studies demonstrate, quite opposing opinions exist regarding a positive correlation between histopathology and reaction to contrast agents, displayed by renal tumors, making sophisticated planning and implementation of further studies necessary.

The time periods during which structures – renal tumor and healthy renal parenchyma - were simultaneously visible in sonography were certainly limiting factors of the current study. The analysis of the images was done by means of Qontrast, Esaote Sp, (EC mark nr. 0051, class IIA). In each image representing the relevant time interval, a total of six areas was selected manually and then analyzed, three in the tumor and three in the healthy renal parenchyma. The size of the selected areas varied in the process, depending on whether the structures to be examined were well visible or not. To minimize the systematic error, only the averages of the relevant measurements were used.

All facts considered, the present study attempted to examine the opposing opinions on which the scientific discussion and argumentation based using detailed differentiation of histological subtypes. Based on the afore-mentioned findings, this study also attempts to suggest a new hypothesis as to how renal tumors react to contrast agents.

5 Conclusions

For both histopathology and the diverse ranges of application, better understanding of CEUS allows new dimension not only for experimental but above all for clinical medicine in everyday life. All these findings confirm the relevance of CEUS as an essential additional diagnostic tool. This relatively new method offers manifold ways of diagnosis and future oncological therapy. Establishing CEUS in clinical routine allows fast, correct, low-risk and cost-effective examinations.

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