Introduction
Bladder cancer (BC) has the highest lifetime treatment cost per patient of all cancers [
1], amounting in the European Union to approximately €4.9 billion per year [
2]. Approximately 75–85% of patients with BC present with the disease confined to the mucosa (stage pTa, pTis) or to the submucosa (stage pT1). Of these patients approximately 50% will have at least one tumor recurrence, and periodic cystoscopies of all these patients are mandatory to detect recurrence as early as possible. Long-term cost benefits can be achieved through reduced tumor recurrence and potentially reduced progression rates [
1]. In addition to recurrence, 11% of initial non-muscle-invasive bladder cancers (NMIBC) will progress to a muscle-invasive stage [
3]. High-grade pT1 (formerly pT1 G3) BC in particular has a high propensity to recur and will progress to a muscle-invasive stage in approximately 50% of patients [
4]. The unpredictable biological behavior of NMIBC, including the development of locally invasive tumor growth, the latter mainly a risk for patients with pT1 tumors, is one of the dilemmas faced during the treatment of the disease. Currently, the search for reliable serum- and urine-based, histological or staging criteria that can adequately predict recurring or progressive tumors is ongoing. Identification of patients at high risk is needed to select them for a more aggressive approach to therapy and follow-up.
There are many strategies to classify the risk of recurrence and/or progression of BC. Generally, NMIBC can be stratified into high-, intermediate, and low-risk groups depending on tumor stage, grade, size, number, and recurrence pattern [
3,
5]. Although the strength of the European Organization for Research and Treatment of Cancer (EORTC) risk groups is their excellent database, in individual cases it is sometimes difficult to decide on the basis of the primary tumor occurrence because of the need for the time-dependent factor “recurrence” and because of the likelihood to have understaging in transurethral resection of bladder tumors (TURBT) [
6]. Technical improvements in the surgical treatment of NMIBC, such as photodynamic diagnosis-assisted transurethral resection of bladder tumor, are targeted at a reduction in recurrence rates [
7]. Many novel tumor markers have been identified and are still being evaluated to improve the accuracy of prognosis and therapy [
8,
9].
Calgranulin A (S100A8) belongs to the S100 protein family, which has more than 20 members, all carrying a Ca
2+-binding EF-Hand motif. Calgranulin A is a protein of 10.83 kD and its gene is located at chromosome 1q21 [
10]. It forms a heterodimer with calgranulin B (S100A9) which is called calprotectin. Calgranulin A may function in the inhibition of casein kinase and as a cytokine, and S100 proteins are involved in cell differentiation and cell cycle regulation [
11,
12]. The precise function of the S100 proteins in tumorigenesis and tumor progression is still unknown but recent reports emphasize their role as a regulator of tumor progression [
13]. Calgranulin A has been identified as highly expressed in muscle-invasive and advanced BC; however, studies of calgranulin A expression and its role in NMIBC are sparse.
The aim of this study was to assess the risks of recurrence and progression dependent on calgranulin A immunostaining in transurethrally resected bladder cancer specimens for the identification of NMIBC at high risk of recurrence or progression.
Discussion
The main predicament of NMIBC remains the high percentage of patients who will suffer recurrence or progression. Patients with pTa tumors have a likelihood of recurrence from 50% to 80%, but on the other hand, the main threat of the pT1 and pTis stages is the risk of progression to a higher stage (10–30%) [
27]. Some patients with pT1G3 BC will undergo lifelong invasive surveillance without any recurrence after therapy [
28]. It is crucial for the outcome of the disease to identify those tumors with high risk of recurrence and progression, in order to adjust the treatment or surveillance strategy according to individual risk. The data most commonly used to predict the further outcome and treatment of BC is currently clinicopathological, such as the tumor stage, tumor grade, size of the tumor, multifocality, and prior recurrence rate. To quantify the risk and implement risk groups, EORTC developed a scoring system based on six variables to calculate the probability of recurrence (score from 0 to 17) and progression (score from 0 to 23), using data from 2596 patients who participated in seven EORTC trials [
3]. On the basis of these scores, the European Association of Urology (EAU) defined risk groups for recurrence and progression in their guidelines [
29]. Different prognostic models and nomograms for risk grouping, using clinicopathological patterns, have been published for NMIBC and have been widely used in clinical practice, starting in 1989 with the British Medical Research Council Subgroup on Superficial Bladder Cancer [
30], later with the Spanish group led by Millan-Rodriguez [
5], and recently by Sylvester’s EORTC risk tables [
3]. In contrast to the use of clinicopathological parameters alone, the nomogram of Shariat et al., which uses age, gender, urine cytology, and a urine-based biomarker test (nuclear matrix protein, NMP22), can predict the probability of recurrence and progression in NMIBC with high accuracy [
31]. Recent findings have demonstrated that NMIBC can be grouped into three major subclasses with basal- and luminal-like characteristics and different clinical outcomes [
32]. Finally, a recent report highlighted the assessment and reporting of lymphovascular invasion and variant histology in TUR specimens as important for risk stratification and decision-making [
33].
In search of novel prognostic tools, a great variety of biomarkers have been examined. For implementation in routine clinical practice, a prognostic marker should be simple and cost-effective, such as the immunostaining of pathological specimens.
Calgranulin A has been shown to be associated with invasive and metastatic tumor growth. The association of the deregulation of S100A8 has also been demonstrated in prostate (sample size 75; immunohistochemistry) [
34], colon (sample size 23 matched pairs; 2D-PAGE and MALDI-MS) [
35], gastric (sample size 218; TMA immunohistochemistry) [
36], and gall bladder cancer (sample size NA; two-dimensional liquid chromatography and tandem mass spectrometry) [
37].
Tolson et al. used the SELDI-TOF analysis of 12 pairwise BC specimens, comparing muscle-invasive BC biopsies with homologous normal tissue, to demonstrate that calgranulin A is highly overexpressed within tumor cells, and suggested a prognostic value of S100A8 in BC [
38]. A systematic evaluation of the S100 protein family, using microarray and real-time PCR in murine and human BC specimens (sample size NA), detected S100A8 overexpression in comparison to normal bladder tissue [
39]. Associations with stage progression, invasion, metastasis, and poor survival have also been presented in a review by Yao et al. [
13]. Pilchowski et al. identified calgranulin A as significantly overexpressed in metastatic BC compared to non-metastatic muscle-invasive BC specimens, via Protein Chip technology surface enhanced laser desorption/ionization time of flight mass spectrometry (sample size 88) [
40]. In a proteomic study from Minami et al. comparing the pre- and postoperative sera and bladder tumor specimens from 77 BC patients, S100A8 was identified as associated with muscle-layer tumor invasion and cancer-specific survival [
41]. Ebbing et al. detected urinary calprotectin concentrations significantly higher in patients with bladder cancer than in healthy controls (sample size 181) [
42]. Finally, a microarray gene expression profiling study by Kim et al., using a four-gene signature including S100A8 gene expression levels, showed that these gene signatures could significantly predict the progression of muscle-invasive bladder tumors (sample size 128) [
43].
There is still sparse information for NMIBC about the usefulness of calgranulin A as a prognostic tool. Ha et al. offered the first evidence for this, examining the mRNA expression of S100A8 in 103 NMIBC tissue samples, and concluding that calgranulin A might be a useful prognostic marker for progression to MIBC [
44]. Kim et al. illustrated that the microarray gene expression profiling of 103 tissue samples is a promising diagnostic tool for the identification of NMIBC patients with a high risk of progression to MIBC [
45]. Bansal et al. detected significant differences in calgranulin A expression between low-grade and high-grade BC in 160 preoperatively examined patients, using serum-based proteomics [
46], and could even show that expression levels were gradually and significantly reduced after surgical treatment [
47].
In the present study, the immunostaining of tumor cells with an antibody against calgranulin A showed significant differences regarding the tumor stage and grade of patient specimens. It therefore appears that its expression positively correlates with tumor invasiveness and differentiation, as demonstrated by previous research groups [
38‐
40]. Remarkably, calgranulin A expression levels in NMIBC, observed via immunostaining, are very low, with 66.5% of all superficial tumor specimens showing no calgranulin A expression at all (calgranulin A staining index = 0). The overexpression of calgranulin A in NMIBC emerged as a potential prognostic factor, however, and Kaplan–Meier estimates showed significant results for recurrence, progression, and CSS. This suggests a prognostic value, as Cox multivariate regression highlighted calgranulin A expression as an independent prognostic factor for recurrence and progression.
This investigation was a preliminary test to translate the increasingly identified usefulness of calgranulin A as a prognostic marker in BC into clinical routine. Immunostaining with calgranulin A could be performed in routine histopathological assessment, to identify patients with NMIBC and a high risk for recurrence and progression.
This study is the first presentation of calgranulin A immunostaining in NMIBC and is a preliminary test because of its limitations: the small number of patients cannot compete with prognostic considerations as derived for instance from the current EORTC risk tables that comprise data from over 2500 patients [
3]. However, calgranulin A showed significant time/event differences with our smaller dataset already, hence follow-up research on this marker should be interesting. Another shortcoming of the work is its retrospective design and the need to calculate the cutoff value retrospectively. Additionally, the quality of our follow-up data may be influenced by the lack of a standardized follow-up protocol: follow-up according to the EAU guidelines was requested, but we could not monitor the reliability of guideline conformity in some cases, when referring urologists did the follow-up themselves. Since the 2004 (and 2010) WHO grading system for urothelial cancers has brought distinct changes compared to the 1973 WHO grading system, which still was used in the study, it will be difficult to compare our data with recent cohorts because of overlapping of G2 grading and low- and high-grade tumor grading, respectively. In this regard, keeping the 1973 WHO grading in pathological reports together with the new grading is important. Since age, gender, and multifocality have been proposed as prognostic factors in the past, we assume that different results than ours, e.g., that age, gender, and multifocality were not constantly significant in Kaplan–Meier survival analysis, may arise with higher patient numbers in a possible validation study. Restricting the Cox proportional hazard analysis to the factors of the EORTC risk tables may influence the results of the multivariate regression, because other factors, e.g., age and gender, have already been identified as prognostically relevant. Because the number of events in our dataset was a limiting factor, this has to be validated in larger cohorts.
Further studies are needed to define the reliability and prognostic value of immunostaining with calgranulin A in NMIBC. These should be based on a larger cohort of patients, at best multicentric, and should be designed with a prospective setting. A comparison of calgranulin A expression, detected through immunostaining, with expression levels assessed by other techniques, e.g., mRNA expression or serum-based proteomics, would consolidate the findings. Before clinical acceptance of calgranulin A as a prognostic marker in NMIBC, a defined follow-up schedule should be executed in a prospective setting to make data suitable for calculating positive and negative predictive values in a time-dependent predictive model.