Prognostic markers in low-grade papillary urothelial neoplasms of the urinary bladder: an update

Prognostic markers in low-grade papillary urothelial neoplasms of the urinary bladder: an update

Review Prognostic markers in lowgrade papillary urothelial neoplasms of the urinary bladder: an update urothelial neoplasms. However, patients with ...

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Review

Prognostic markers in lowgrade papillary urothelial neoplasms of the urinary bladder: an update

urothelial neoplasms. However, patients with low-grade tumours present, in general, a high risk of recurrent disease and certain possibilities to progress either in grade or in stage when longterm follow-up programmes are performed.2 From a histopathological point of view, in papillary urothelial bladder neoplasms the assessment of histological grade and tumour stage by the pathologist is mandatory for the adequate management of patients. Based on both histological and image analysis studies, a recent 1998 World Health Organization/International Society of Urologic Pathologists (WHO/ISUP) consensus classification and 1999 WHO classifications were proposed for malignancy grading of papillary urothelial neoplasms of the urinary bladder. These grading systems were designed in an attempt to clarify the different categories of bladder tumours associated with a different biological behaviour.3,4 Indeed, two major categories of bladder neoplasms were initially distinguished: lowgrade papillary urothelial neoplasms (LGPUN) and high-grade papillary urothelial neoplasms (HGPUN). Additionally, a new category of low-grade neoplasms in which an architectural variation of tumour cells was not readily seen was also considered. This new category did not exist in previous classifications and was referred to as papillary urothelial neoplasms of low malignant potential (PUNLMP). Interestingly, this new category does not carry the uncomfortable label of carcinoma. In the WHO 1973 classification system, PUNLMP and LGPUC were both graded as G1 (grade 1) papillary urothelial carcinomas, including a small subgroup with the best differentiated G2 (grade 2) papillary urothelial carcinomas as well.5 Importantly, the vast majority, if not all, of these lesions are non-invasive neoplasms.6,7 For the specific subgroup of LGPUN, remarkable differences in terms of recurrence rate and tumour progression have been reported when patients are followed up long-term.8–10 In contrast, other series have not demonstrated such a prognostic distinction between PUNLMP and LGPUN.11,12 Furthermore, other investigations describe that the cytological detection of papillary urothelial carcinoma from bladder washings does not improve with the establishment of the 1998 WHO/ISUP classification system.13 Nevertheless, the assessment of the histological grade by urological pathologists is a subjective decision and, frequently, the application of morphological criteria is complicated due to the intra- and inter-tumour heterogeneity, and to the existence of a varying number of borderline cases situated in consecutive groups.12 This review examines the current controversy regarding the assessment of histological grade in papillary bladder tumours, as well as a series of clinicopathological, morphometric, cytometric, immunohistochemical and molecular markers which have been reported as useful indicators of the clinical course of patients with non-invasive but highly recurrent LGPUN (PUNLMP and LGPUC) of the urinary bladder.

David Ramos

Abstract Papillary urothelial neoplasms of the urinary bladder comprise a heterogeneous spectrum of ‘continuous’ lesions in which the assessment of an accurate histological grade and tumour stage is mandatory for the clinical management of patients. The 1998 World Health Organization/International Society of Urologic Pathologists (WHO/ISUP) consensus classification and the 1999 WHO classification proposed new malignancy grading schemes, mainly based on morphometric studies for the replacement of the 1973 WHO grading system. In accordance with these novel grading systems, two major categories of papillary urothelial neoplasms were distinguished: low-grade and high-grade papillary urothelial neoplasms. Concerning the specific subgroup of low-grade tumours, two other entities were defined: papillary urothelial neoplasms of low malignant potential (PUNLMP) and low-grade papillary urothelial carcinomas (LGPUC). In long-term follow-up programmes, PUNLMP have demonstrated low recurrence rates and minimal risk for tumour progression in comparison with LGPUC. However, grade assessment is a subjective decision and, on occasion, the use of reproducible criteria by urological pathologists is difficult due to tumour heterogeneity and to the existence of a variable number of cases situated between consecutive groups. As a result, to determine a better correlation with clinicopathological patient outcome, other predictive markers are being investigated. Among these, the prognostic significance of classical clinicopathological, morphometric, cytometric, immunohistochemical and molecular markers have been widely reported. This review summarizes the prognostic importance of all these markers in the prediction of tumour recurrences and progression in lowgrade papillary urothelial bladder neoplasms.

Keywords bladder; neoplasms; prognostic markers; pathology

Introduction Tumours of the urinary bladder are the second most prevalent urological tumours in men after prostate carcinomas, and the fourth or fifth in mortality rate.1 Within these tumours, papillary urothelial neoplasms account for the most common histological type and, approximately, 80% are ‘superficial’ neoplasms without invasion or with infiltration of the lamina propria. The favourable clinical course of patients with urinary bladder tumours is determined not only by the diagnosis in the early phases of tumour development but also by the high percentage of low-grade and non-invasive or minimally invasive papillary

Classical clinicopathological markers: grade assessment controversy In addition to tumour stage, histological grade also plays a pivotal role in the clinical management and outcome of patients with papillary urothelial tumours of the urinary bladder. As above mentioned, the WHO 1973 classification system considered three different categories for papillary urothelial neoplasms (WHO grades 1, 2 and 3). Recently, the WHO/ISUP 1998 and WHO 1999 ­malignancy grading

David Ramos MD is Professor at the Department of Pathology, School of Medicine, University of Valencia, Valencia, Spain.

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systems proposed a similar distribution of histological grades but with some remarkable changes overall in LGPUN.3,4 In these malignancy grading systems, PUNLMP appeared as a new entity of low aggressive lesions encompassing the most well-differentiated of WHO 1973 G1 carcinomas. Meanwhile, LGPUC included the remaining G1 carcinomas of the WHO 1973 system together with a small subgroup of the most well-differentiated within the broad spectrum of G2 carcinomas. Moreover, remarkable prognostic differences between LGPUN and HGPUN have been statistically demonstrated in some recent publications.11,14–17 However, the predictive value and diagnostic reproducibility between PUNLMP and LGPUC have not been homogeneously corroborated in some of these series.11,17 Given the strong resemblance of PUNLMP and LGPUC, the most objective histological finding for their distinction might be, in our opinion, the presence of mitotic figures in basal cells in PUNLMP or in the apical layer in LGPUC (Figure 1). Bircan et al. suggested that the WHO 1999 system may be more useful to evaluate bladder tumors histopathologically in comparison to the WHO 1973 and WHO/ISUP 1998 systems.18 In contrast, Bostwick et al. recently described that the WHO 1973 classification is probably superior in terms of ­ prognostic ­correlation to the recently proposed WHO/ISUP 1998 and WHO 1999 malignancy grading systems.19

Cheng et al. considered that tumour grade assessment should take into consideration cancer heterogeneity.20 In this regard, they proposed the use of a combined score based on both most extensive and worst histological grades: thus, the combined grade ranged from 2 to 6. Moreover, they also demonstrated that this system presented a strong statistical correlation with patient outcome.20 When considering the simultaneous utilization of certain immunohistochemical markers, Yin and Leong observed that tumour recurrences correlate much better with papillary lesions classified by the 1998 WHO/ISUP system than with the 1973 WHO system.21 Regardless of tumour grade and stage, other classical markers have also been investigated in an attempt to predict more accurately the clinical course of patients with LGPUN (PUNLMP vs LGPUC). These classical markers include tumour size and multiplicity, which have been widely reported in the literature as showing prognostic correlation in predicting recurrences and tumour progression either in grade or in stage in the specific subgroup of LGPUN of the urinary bladder.22–24 Nevertheless, tumour grade and stage, along with other morphometric and immunohistochemical markers, have proven to be, in general, better correlated with prognosis than tumour size and multiplicity.

Morphometric and cytometric markers Based on the image analysis of histological tumour sections, morphometric studies were the basis for the objective definition of tumour categories in the WHO/ISUP 1998 consensus classification and the WHO 1999 malignancy grading systems. Herein, different groups of papillary urothelial bladder neoplasms were defined according to the degree of architectural variation, as well as the deviation of the nuclear shape of the tumour cells.3,4,15,25 Regardless of these systems, the prognostic importance of morphometric factors, taken independently, such as mean nuclear area (MNA) and standard deviation (SDMNA), mean nuclear perimeter (MNP) and standard deviation (SDMNP), and roundness factors (RF) has been widely reported in the literature.22,26–29 A great intra- and inter-tumour heterogeneity of tumour cells was clearly noted in these series, and a varying number of elements of interest (nuclei of tumour cells) were subsequently measured in the most atypical areas of the tumour in previously selected histological sections. Indeed, some authors examined in their series a total of 100–300 nuclei from the inner and outer layers of the tumour urothelium, while others examined only the 10–50 largest nuclei independently of their position within the papilla cell layers.23,26–29 In these studies, the superficial ‘umbrella’ cells were systematically not taken into account. We have recently described, in agreement with Bol et al., an excellent prognostic correlation with the MNA of the 10 largest nuclei in the specific subgroup of LGPUN,22,30 although many other morphometric factors were also shown to be strongly correlated in univariate studies of recurrence prediction and relapse-free interval (RFI) in LGPUN. This finding highlighted the influence of tumour cell heterogeneity on the morphological identification of atypical clones of cells in these low-aggressive lesions. In close accordance with morphometric data, the assessment of DNA content by image cytometry (ICM) has also proven to be of considerable prognostic significance in LGPUN.31 In this regard, we have recently corroborated, in addition to the MNA of the 10 largest nuclei, the value of DNA content by ICM of the 10 largest nuclei in the prediction of tumour recurrences and RFI

a Histological appearance of a papillary urothelial neoplasm of low malignant potential (PUNLMP) and b a low-grade papillary urothelial carcinoma (LGPUC), highlighting the different localization of mitotic figures in basal cells in PUNLMP and apical cells in LGPUC (haematoxylin & eosin). Figure 1

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in LGPUN (unpublished observation). An important advantage of measuring the DNA content of tumour cells by ICM instead of flow cytometry (FCM) is the objective and specific selection of the most atypical objects of interest, directly observed under the microscope on previously selected histological sections of the tumour. Importantly, FCM does not permit a correct discrimination of small populations of tumour cells, which can be essential in LGPUN for a correct discrimination between PUNLMP and LGPUC, and subsequently for an adequate prediction of the clinical outcome and management of patients.32

Immunohistochemical versus molecular methodologies Immunohistochemistry consists of a multistep and relatively complex laboratory procedure, which involves multiple internal and external factors that can influence the correct interpretation and reproducibility of the results. These influencing factors include, for example, the utilization of polyclonal or monoclonal antibodies, antigen retrieval methods, dilutions, reagents, temperature, pH, frozen or formalin-fixed and paraffin-embedded material. Therefore, for a more consistent interpretation of data, as well as for an adequate reproducibility of the methodology across different laboratories, standardization of the immunohistochemical techniques is mandatory in order to achieve adequate and universally reproducible prognostic markers. In addition, an important advantage of immunohistochemistry in comparison with other novel molecular methodologies is that the latter technologies are much more complex, sophisticated and costly than the former. Nevertheless, the rational use of both immunohistochemical and molecular technologies is currently essential in the search for bladder cancer prognosticators.

a, b Low versus high cell proliferation index (Ki67 labeling index) in low grade papillary urothelial neoplasms (immunohistochemistry). Figure 2

Cell proliferation status

clones of cells when, at the morphometric level, the MNA of the 10 largest nuclei were measured. In this same investigation, these two variables (Ki67 antigen immunoexpression and the MNA of the 10 largest nuclei) both appeared to be independent predictors of recurrent disease in multivariate statistical studies.22 Similarly, novel markers are being investigated concerning the immunohistochemical assessment of tumour cell proliferation. In particular, human DNA topoisomerase-IIalpha expression has demonstrated a strong prognostic correlation in predicting tumour recurrences and progression in papillary bladder neoplasms.42

In addition to the classical assessment of mitotic activity, more accurate methods have been utilized by pathologists for the specific evaluation of tumour cell proliferation in papillary urothelial neoplasms of the urinary bladder. Of these methods, the immunohistochemical assessment of PCNA antigen expression and, currently, the wide utilization of Ki67 (MIB1) antigen expression, frequently referred to as Ki67 labelling index (Ki67 LI), have been the most commonly used cell proliferation indexes. Ki67 antigen is exclusively expressed by proliferative cells in any phase of the cell cycle (G1, S-phase, G2 and mitosis). The prognostic importance of cell proliferation status as determined by immunohistochemical assessment of Ki67 LI has been widely studied in papillary urothelial bladder neoplasms of any grade and stage,33–36 although others have not demonstrated such a prognostic significance in their series.37–39 However, some other studies with longterm follow-up of patients have statistically proven an independent prognostic correlation of this proliferation cell marker regarding the prediction of recurrent and progressing disease.33,40,41 In non-invasive LGPUN, we have described, in agreement with Pfister et al.,41 a strong statistical correlation between the maximum Ki67 LI and the prediction of tumour recurrences or RFI (Figure 2).22 However, the cut-off values established for this proliferative marker have been quite variable in the different series. In our study, we noted, as did Bol et al.,30 that LGPUN with a high proliferation status also showed the most atypical

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Cell cycle regulation Progression through G1 to S-phase is mainly controlled by cyclindependent kinases (CDK) 4 and 6, which bind specifically to the D-type cyclins (D1–D3), forming complexes that phosphorylate to the product of the RB gene (pRb). Phosphorylation of pRb releases the E2F transcription factors implicated in preparing the nucleus for DNA replication and forcing cells to enter into mitosis. In turn, CDK activity is influenced by CDK inhibitors (CDKI) that physically associate with cyclins, CDK or their complexes. Among these CDKI are p16INK4A and p15INK4B, which form binary complexes exclusively with the CDK4 and CDK6, inhibiting their function and, by so doing, inhibit pRb phosphorylation during G1 phase. INK4A and INK4B genes map to chromosome 9p21, where they are found in tandem. The INK4B gene is composed of two exons, whereas 44

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cyclin E has specifically demonstrated a strong statistical correlation with a poor prognosis from either positive or negative results. Recently, Santos et al. studied the immunohistochemical expression of p16, p27, pRb, p53 and Ki67 labelling index in LGPUN and concluded that only Ki67 antigen expression was a reliable marker of recurrent disease.60 Similarly, Pfister et al. found that cell cycle regulators (p16, p27, p53 and pRb) did not present prognostic correlation in superficial bladder carcinomas of all histological grades, whereas Ki67 LI again appeared to be the best indicator of recurrent-free and progression-free survival.41 Shariat et al. described that, interestingly, combinations of p53, pRB, p21 and p27 had cooperative/synergistic effects, stratifying patients into different risk groups regarding recurrent or progressive bladder diseases.61 In summary, studies encompassing large series of patients and long-term follow-up are needed for the complete evaluation of LGPUN of the urinary bladder, by corroborating with certainty the prognostic importance of the broad spectrum of cell cycle regulation proteins and their respective genes at both immunohistochemical and molecular levels.

the INK4A gene contains three exons and exhibits two distinct transcripts derived from two alternative first exons, exon 1α and exon 1β. The p16INK4A protein is composed by the α transcript, and the alternatively spliced β transcript encodes the p14ARF ­protein, which is a potent regulator of the cell cycle, functioning in a manner different from the CDKI. P14ARF acts upstream in the p53 pathway by binding to MDM2 and preventing p53 degradation, and thus permitting p53-induced apoptosis or growth arrest.43 The TP53 gene is a tumour suppressor gene specifically located on the short arm of chromosome 17. This gene negatively regulates cell cycle progression by arresting cell division in response to antimitogenic or stress signals, such as after DNA damage. The phenotypic expression of the TP53 gene is the wild-type p53 protein. Mutations in this gene produce an abnormal non-functional p53 protein with a prolonged half-life in comparison to the wildtype protein. As a result, this abnormal expression of p53 can be readily detected in tumour cell nuclei by immunohistochemical methods. A strong statistical correlation between p53 immunoexpression and the detection of TP53 mutations at the molecular level has been described in diverse studies.44,45 In this context, in patients with advanced invasive bladder carcinomas, the response to adjuvant chemotherapy was only associated with the immunohistochemical and molecular assessment of p53 overexpression.46 In superficial bladder neoplasms, which include papillary urothelial neoplasms of all histological grades, the immunohistochemical expression of p53 has also shown prognostic significance with recurrent and progressive disease.47,48 In other series with superficial bladder tumours, an independent prognostic significance in multivariate statistical studies was also demonstrated.49,50 Cassetta et al., in their series of non-invasive LGPUN, also described an independent prognostic significance for p53 overexpression regarding recurrence prediction.51 Likewise, our group and other investigators52,53 have also corroborated the prognostic importance of p53 immunoexpression as an excellent indicator of tumour recurrences in the specific subgroup of LGPUN. Similarly to Ki67 antigen expression, the cut-off values for p53 were, at the immunohistochemical level, quite variable in the different series (range 0–30%), and this would also need to be standardized. Other cell cycle regulators have also been investigated at the immunohistochemical level as possible predictive indicators of the clinical course of patients with papillary urothelial bladder neoplasms. The most common molecular alteration in the early phases of bladder carcinogenesis is the loss of the short arm of chromosome 9 (9p21 locus), where the CDKN2A/ARF locus gene is located. This locus encodes for two different proteins, p14ARF and p16INK4A, which are known to be directly implicated in the regulation of the pRb and p53 pathways. Other proteins encoded by different genes on different chromosomes also contribute to the negative regulation of the cell cycle, such as the KIP family of proteins (p21 and p27) or pRb (retinoblastoma gene). In contrast, MDM2, cyclin D1 and cyclin E are normally overexpressed in high-grade, high-stage urothelial neoplasms involved in the final phases of bladder tumourigenesis.54–59 Nevertheless, the prognostic importance of many of these cell cycle regulation genes has not been confirmed and, commonly, the immunohistochemical expression of cell cycle regulation proteins is aberrant and quite different from the expected values observed in the molecular analyses of their respective genes. In some of the above-mentioned series, the expression of p27 and

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Apoptosis The bcl-2 gene was first described in follicular lymphomas as an anti-apoptotic gene identified on the long arm of chromosome 18 (18q21). Apoptosis can be defined as a genetically programmed cell death, regulated by diverse promoting genes such as tumour necrosis factor (TNF), TP53, Ras, Bax and Myc; and anti-apoptotic genes, such as the bcl-2 gene. In papillary urothelial neoplasms of the urinary bladder, the immunohistochemical expression of bcl-2 has been the most extensively studied phenotypic alteration and has generally shown a specific basal cell distribution in the normal non-tumoural urothelium as well as in many LGPUN. In contrast, as tumour grade and stage increase, the distribution of bcl-2 immunostaining tends to be located in intermediate and superficial cells of the tumour urothelium. However, the prognostic significance of bcl-2 overexpression has varied between the different series. Some authors have described a strong prognostic correlation between bcl-2 overexpression and a worse clinical course for patients,62 whereas others have reported a less aggressive biological behaviour.63 In the specific subgroup of non-invasive LGPUN, an excellent statistical association between bcl-2 overexpression and the prediction of recurrent disease has been described,53 although other series have not confirmed this point.36,64 Vollmer et al. analysed the dual immunohistochemical expression of bcl-2 and p53, and observed that a diffuse bcl-2 immunoexpression through all urothelial layers, along with p53 negativity, determined an excellent clinical course for patients, whereas p53 overexpression, independent of bcl-2 status, implied in general a worse clinical outcome.65 We have recently described similar results for p53 and bcl-2 immunoexpression in a series of 120 patients with non-invasive LGPUN. However, the number of cases showing this pattern of non-basal cell bcl-2 staining, along with p53 negativity, was not high enough in our series to be of prognostic significance at the statistical level.53 Karam et al. reported that, immunohistochemically, the assessment of a combined apoptotic status by means of diverse altered biomarkers in patients treated by radical cystectomy provides prognostic information that could help to identify those patients at risk for disease recurrence and mortality, and who could ­benefit 45

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from early adjuvant treatment.66 However, it remains unclear if this observation applies to low-grade bladder tumours as well.

studies comprising larger series of patients with long-term followup are needed in order to select which patients could probably benefit from spacing control cystoscopies. Additionally, CK20 immunostaining has been demonstrated to be an important diagnostic biomarker that can be used in routine practice for the identification of urothelial bladder lesions in urine cytology smears.76 Therefore, in our opinion, an abnormal cytokeratin 20 or 34betaE12 antigen expression in low-grade papillary urothelial neoplasms, when compared with the normal urothelium, should be taken into account in routine practice to estimate the risk of tumour recurrences and even tumour progression (Figure 3 and Figure 4).

Oncogenes The erbB-2 (c-neu/Her2-neu) gene is a proto-oncogene specifically located on the short arm of chromosome 17 (17p21). ErbB-2 amplification has been observed in a high percentage of breast, ovarian, lung and gastric carcinomas. The results of studies of c-erbB-2 amplification in papillary urothelial neoplasms of the urinary bladder are controversial. At the immunohistochemical level, McCann et al. described an overexpression of c-erbB-2 in up to 17% of breast cancers, but in only approximately 2% of papillary urothelial bladder neoplasms.67 Other studies have also demonstrated c-erbB-2 overexpression in a high percentage of HGPUN,68 although many others have not corroborated such a prognostic correlation in identical series of tumours.69 In these studies, only a small percentage of LGPUN showed c-erbB-2 overexpression. In LGPUN, we encountered a relatively high percentage of c-erbB-2 immunostaining in comparison with these other studies, although we did not find a strong statistical association with recurrence prediction.52 EGF-R (epidermal growth factor receptor) is a transmembrane protein encoded by the c-erbB-1 gene. This protein is specifically expressed by the basal cells in the normal urothelium of the bladder. Alterations on this pattern has been reported as having prognostic correlation and to be indicative of a high risk of recurrent and progressing disease in superficial carcinomas of all histological grades.70 However, to our knowledge, specific studies regarding the prediction of tumour recurrences and relapse-free interval in non-invasive LGPUN have not been performed to date.

Cytokeratin expression patterns The cytokeratin family comprises a broad spectrum of at least 20 different cytoplasmic intermediate filaments, which are expressed by a high number of epithelial non-tumoural cells and their malignant counterparts. In urinary bladder lesions, diverse cytokeratin epitopes have been immunohistochemically identified in the different cell layers of the normal non-tumoural urothelium. Herein, CK20 is normally expressed by superficial and some isolated intermediate cells, whereas 34βE12 antigen (high-molecular weight cytokeratins) is generally limited to the basal cell layers of the urothelium.71–74 In papillary urothelial bladder neoplasms, these cytokeratin expression patterns may be maintained, lost or altered as the tumour grade and stage increase, which could be indicative of the patient outcome in relation to recurrence prediction and relapse-free interval. Our group recently described that, independently of the administration of adjuvant local chemotherapy, the immunohistochemical assessment of a normal cytokeratin expression pattern with CK20 in superficial cells, along with a basal cell distribution with 34βE12 antigen, were statistically associated with an excellent clinical outcome for patients, in comparison with a worse clinical course when an abnormal staining pattern with either of these two cytokeratins was observed.65 In this study, we also determined a better statistical correlation with recurrence prediction and RFI when a evaluation of the topographic immunostaining pattern with both cytokeratins was performed simultaneously.75 To confirm all these findings, further

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a Normal urothelium obtained from a non-tumour patient showing b the characteristic cytokeratin expression patterns of cytokeratin 34betaE12 antigen and c cytokeratin 20 (haematoxylin & eosin). Figure 3

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a Low-grade papillary urothelial neoplasms (haematoxylin & eosin). b,c Note the maintenance of normal cytokeratin expression patterns or d the appearance of abnormal staining patterns, usually in a diffuse manner, with both cytokeratin 20 and 34betaE12 (immunohistochemistry). Figure 4

with tumour cell initiation, followed by the specific selection of the most aggressive and most atypical clones of cells for eventual progression to muscle-invasive HGPUN.79 As mentioned above regarding the tumour phenotype, the molecular changes found in LGPUN are also heterogeneous and, therefore, the accurate utilization of microdissection techniques on the tumour samples prior to the application of novel molecular and immunohistochemical methodologies would be of great interest.80,81 In the specific subgroup of LGPUN, the initial genetic event identified by cytogenetics, loss of heterozygosity (LOH) and other PCR-based methodologies is the loss of genetic material at chromosome 9. In this regard, Hartmann et al. provided evidence that deletions in the short arm of chromosome 9 (9p21) appear to precede deletions found in the long arm of the same chromosome (9q).77 On the other hand, other genetic alterations in genes such as NF-1, TP53, RB-1, WT-1 and C-erbB-2 have frequently been observed in HGPUN originating directly from the bladder urothelium or from the ­biological transformation of previous LGPUN.79 Nevertheless, most of these molecular findings (tumour genotype) have not been subsequently corroborated at the immunohistochemical level (tumour phenotype). By comparative genomic hybridization, LGPUN have also shown very similar genetic abnormalities, with specific losses of genetic material at chromosomes 9p (CDKN2 gene), 9q and Y; gains at chromosomes 1q and 17q (TP53 gene); and amplification at chromosome 11q (cyclin D1).80

Molecular markers In the last few years the exponential accumulation of molecular findings has elucidated new and interesting data regarding the natural history of bladder carcinogenesis. In fact, diverse molecular studies have demonstrated that multifocal papillary urothelial neoplasms of the urinary bladder are probably monoclonal in origin, and, as a result, these lesions might be derived from the same progenitor cell.77,78 Biologically, urothelial bladder neoplasms can be classified into three different categories: LGPUN, which are usually superficial lesions without stromal invasion or with infiltration only of the lamina propria; HGPUN, which are usually deep or muscle-invasive carcinomas; and flat urothelial lesions, such as urothelial dysplasias and urothelial carcinoma in situ, both of which are frequently associated with HGPUN at clinical presentation. LGPUN have, in general, a good clinical course, although these low-grade lesions can progress to HGPUN through the progressive accumulation of different genetic abnormalities (multistep carcinogenetic process). These molecular alterations mainly affect the control of tumour cell proliferation (cell cycle regulation checkpoints) and tumour cell senescence (apoptosis), both of which can be considered as the key factors in the natural history of bladder tumourigenesis.79 Thus, LGPUN usually appear as multiple non-invasive lesions at different times and at different locations in the bladder urothelium. These lowgrade lesions usually evolve in a multistep process consistent

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The CDKN2A/ARF locus comprises two different tumour suppressor genes located at the locus 9p21. These genes encode for the CDK inhibitors p14 and p16, although other negative cell cycle regulators such as p15, p21, p27 could also be implicated in the early phases of bladder carcinogenesis. The inactivation of the CDKN2 gene is involved in the genetic alteration of RB-1 and TP53 molecular pathways in relation to progressive bladder disease.82,83 Indeed, specific molecular alterations in RB-1 and TP53 are frequently encountered in HGPUN, as well as in a variable number of LGPUN. Therefore, in our opinion, a pivotal goal in defining the natural history of LGPUN of the urinary bladder would be to check, at the molecular and immunohistochemical level, the key genes and their encoded regulating proteins involved in the G1 to S-phase checkpoint control. In summary, many biological markers have been reported to date with variable prognostic importance in low-grade papillary urothelial neoplasms of the urinary bladder. Of these markers, the paramount importance should be emphasized in the immunohistochemical assessment of cell proliferation status (Ki67 labelling index), along with the assessment of an abnormal cytokeratin immunoexpression pattern with CK20 and 34βE12 antigen. Likewise, the identification of an atypical clone of cells by image analysis or under the light microscope seems also to be just as important. However, further studies mainly based on tissue and genomic array methodologies would be necessary in order to evaluate the existence of more accurate prognosticators of recurrent disease for this group of non-invasive but highly recurrent low-grade bladder neoplasms. ◆

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Practice points • LGPUN of the urinary bladder comprise a heterogeneous and controversial group of non-invasive lesions in which remarkable differences in terms of disease recurrence have been reported in the literature • Different grading schemes have been historically utilized in papillary urothelial tumours of the urinary bladder, seeking the most accurate and reproducible system • In addition to classical clinicopathological factors, diverse biological prognosticators such as morphometric, cytometric, immunohistochemical and molecular markers are currently being investigated in an attempt to clarify the clinical outcome of these patients • The immunohistochemical assessment of cell proliferation status (Ki67 labelling index), along with alterations in cytokeratin expression patterns and the identification of atypical cells, appear to be of paramount importance in predicting patient outcome

Research directions The natural history of LGPUN of the urinary bladder is closely related to genetic and molecular profiles, especially those related to cell cycle progression. In this regard, several attempts have been made to find individual markers that could improve the clinical management of this group of patients. Thus, with regard to the specific subgroup of low-grade papillary urothelial bladder neoplasms, future research will probably be directed towards the identification and characterization of a phenotypic or gene expression signature that could identify those tumours with a more aggressive behaviour, which could be to the benefit of individualizing treatment.

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