Key points

Screening, diagnosis, and evaluation in bladder cancer screening

Bladder cancer (BCa) is a heterogeneous disease with a variable natural history. Most patients (70%) present with superficial tumors (stages Ta, T1, or carcinoma in situ). However, 3 out of 10 patients present with muscle-invasive disease (T2–4) with a high risk of death from distant metastases. Moreover, roughly between 50% and 70% of superficial tumors do recur, and approximately 10% to 20% of them progress to muscle-invasive disease. However, BCa has a relatively low ratio of mortality versus incidence of new cases. In consequence, there is the danger of overdiagnosis and overtreatment.

Hence, the goal and challenge of screening would be to detect the disease at an earlier stage, consequently improving morbidity and survival, but, more importantly, to be able to identify the tumors that are more likely to become muscle-invasive cancers. Such early detection of tumors could allow for earlier curative intervention and could potentially preclude the need for unnecessary surgical treatment or chemotherapy and lower the costs associated with treatment.

To date, an estimated 75,000 new cases of urinary BCa will be diagnosed in the United States (56,390 men and 18,300 women) in 2014. In the same year, approximately 16,000 new deaths (11,170 men and 4410 women) are expected. BCa is the fourth most common cancer and is 3 times more common in men than in women in the United States. It has also been previously reported that the age-adjusted incidence of BCa seems to be increasing over time: from 21.0 to 25.5 per 100,000 person-years between 1973 and 2009 (+0.2% per year, P = .001).

Two landmark studies have evaluated the effect of screening for BCa. Messing and colleagues performed an important assessment based on 1575 cases (≥50-year-old men screened at home using hematuria dipsticks) and 509 controls (nonscreened). Those who showed positive results underwent cystoscopy (n = 283), and 21 (7.4%) of them were diagnosed with BCa. The primary results of that study indicated that earlier detection of BCa could result in a lower proportion of invasive cancers among high-grade and/or muscle-invasive diseases in screened versus nonscreened men (10% vs 60%, P = .002) and a significant reduction in mortality caused by the disease (0% vs 20%, P = .02). Britton and colleagues examined 2356 men aged 60 years and older for dipstick hematuria. The test was positive in 20% of men, and BCa was ultimately diagnosed in 17 individuals (5.3%). Of those, 9 patients had high-risk non–muscle-invasive BCa. At a 7-year follow-up, 5 out of 9 patients progressed to muscle-invasive disease and 3 out of 9 died of the disease.

Partly owning to an overall low incidence of the disease (25.5 per 100,000 person-years in 2009), screening for BCa is currently not recommended as a standard of care during routine clinical practice. The challenge being that a clearly defined high-risk population needs to be identified, so as to avoid the usual harms in screening, such as unnecessary diagnostic-related treatments (ie, cystoscopy and biopsy, transurethral resection of bladder tumor [TURBT], intravesical chemotherapy), and overdiagnosis. Wu and colleagues generated a model based on large case-control data (n = 678 cases and n = 678 controls) for the prediction of BCa risk using established risk factors, such as smoking and well-known occupational exposure (eg, diesel, aromatic amines, dry cleaning fluids, radioactive materials, arsenic). At internal validation, the model demonstrated an area under the curve of 80%. However, the model is impeded by the lack of external validation.

Similarly, Vickers and colleagues using data from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial attempted to create a risk score in order to identify those at higher risk of developing BCa. The study comprised 49,873 persons for the training set and 99,746 individuals for the external validation set. The investigators showed that the trade-off between the number of patients screened and invasive/high-grade tumors avoided was more optimal when restricting screening to a high-risk population instead of the whole population (57 vs 38 per 100,000), hence supporting the strategy to screen a high-risk population only.

Although large-scale screening programs may seem beneficial and critical, the US Preventive Services Task Force has deemed existing evidence to be insufficient in assessing the balance of benefits and harms of screening. Experts have suggested that improvements have to be made with regard to the identification of what should be defined as high-risk populations before a randomized-controlled trial could be designed to validate the hypothesis of an improvement in screening. It is noteworthy that in recent years, little progress in improving survival and reducing mortality for patients with muscle-invasive BCa has been seen. Indeed, despite well-known risk factors (ie, smoking, occupational exposure), as well as the developments in BCa management over time, it did not lead to better cancer control outcomes. In consequence, it may be important to adequately address the challenges of screening in BCa in the near future.

Future considerations for screening

Many unknowns remain before a large-scale program of BCa screening may be put forward. For one, few studies have examined the natural history of screened early stage untreated BCa. The appreciation of how such tumors behave may assist clinicians in understanding the effects of screening and overdiagnosis/overtreatment. Other randomized studies with large sample sizes that assess urine tests and the incidence of BCa, and staging, with follow-up information on clinical and cancer control outcomes are needed. A uniform definition of what constitutes a high-risk population is necessary in order to reduce the potential harms associated with overdiagnosis/overtreatment. Moreover, it may be important to invest in less invasive and costly diagnostic tools but equally sensitive and specific. For example, traditional cystoscopy may not be adequate if population-based screening is envisaged, as the procedure is invasive and not cost-efficient. In order to reduce the number cystoscopies required to diagnose one patient with BCa, the combination of dipstick testing and molecular markers have been suggested. It may also be worthwhile to test the effect of less toxic treatments in patients diagnosed in the earlier stages to prevent progression versus more invasive and toxic treatments reserved for patients diagnosed in the more advanced stages. This testing may be useful in evaluating the trade-offs and costs associated with earlier treatment modalities.

Presentation and investigation

The most common presenting symptom of BCa is gross hematuria. In patients with carcinoma in situ, additional unexplained urinary frequency, urgency, or irritative voiding symptoms may indicate the possibility of BCa. The prevalence of BCa ranges between 13% and 35% in patients presenting with gross hematuria and between 5% and 10% in patients presenting with microscopic hematuria. The proportion of patients with asymptomatic hematuria ranges between 0.2% and 21.0%, when a direct relationship with age has been shown.

The current practice includes urine dipstick or microscopic urinalysis for hematuria, urine cytology, and other tests for urine biomarkers, although their value has not yet been established. Patients with positive results are referred for further evaluation, which generally include cystoscopy, followed by biopsy if necessary, and radiological investigation. Imaging is important in the evaluation for patients presenting with hematuria, primarily to visualize the upper urinary tract, as cystoscopy inspects the lower tracts. Options for imaging include ultrasonography, intravenous urogram (IVU), computed tomography (CT) urography, MRI, or a combination of these.

Bladder tumor markers

Urine cytology, a simple and noninvasive procedure, in combination with cystoscopy is superior to cystoscopy alone in identifying high-grade lesions (ie, carcinoma in situ). It has a good sensitivity for detecting high-grade BCa but a poor sensitivity for detecting low-grade tumors (between 7%–17%). In recent years, several novel developments of urine markers for the detection of BCa have been investigated ( Table 1 ). For example, of 79 patients with BCa, the nuclear matrix protein 22 (NMP-22) assay was positive in 44 patients (sensitivity 56% and specificity 86%). Similarly, fluorescence in situ hybridization (FISH, Vysis Inc., Downers Grove, IL) (ie, UroVysion assay designed to detect aneuploidy for chromosomes 3, 7, 17 and loss of the 9p21 locus via FISH) demonstrated a promising range of sensitivity (70%–86%) and specificity (66%–93%) in a pooled analysis of 4 articles as part of a meta-analysis.

However, urine cytology remains superior in specificity compared with most other markers (up to 98%, see Table 1 ). Moreover, no biomarker has demonstrated superior clinical utility over cytology and cystoscopy combined. Hence, experts agree that it is highly unlikely that a new marker could achieve the accuracy of what cytology and cystoscopy can offer. Currently, the use of urine cytology is recommended by national guidelines despite the paucity of randomized trials that actually examined its use in clinical practice. For patients with noninvasive disease, urine cytology also represents an important surveillance tool.

Perspectives on screening

The current recommendations do not support routine BCa screening because of insufficient evidence and lack of understanding of the effects of screening in the case of overdiagnosis and overtreatment. However, the results of existing studies suggest that BCa screening may be important in high-risk populations. The challenge remains that experts need to be able to diagnose patients at an early stage of the disease and, at the same time, identify the tumors that are likely to progress to muscle-invasive disease. Currently, the combination of urine cytology and cystoscopy remains the gold standard for diagnosing patients with BCa. Less invasive urine biomarkers have been investigated over time, but their performance remains subpar with respect to specificity compared with cytology alone. It is unlikely that a new marker will be used to replace the conventional urine cytology and cystoscopy.

Endoscopic examination of the lower urinary tract

The cornerstone of the diagnosis and subsequent management of BCa is the cystoscopic examination of the lower urinary tract. Specifically, white light cystoscopy (WLC) remains the gold standard, despite its limitations. Recently, new optical diagnostic methods have been designed to improve the accuracy of WLC: fluorescence cystoscopy, narrow-band imaging (NBI), and optical coherence tomography (OCT).

White light cystoscopy

WLC allows examination of the urethra and the bladder. It can be performed with either rigid or flexible endoscopes, depending on the clinical scenario. For screening and diagnostic office-based purposes, flexible cystoscopy is typically used. Although it is a more comfortable instrument than the rigid cystoscope, it has the disadvantages of having a small irrigation port and lacks a separate working sheath. It is also more costly and inclined to failure.

The sensitivity and specificity of WLC range from 62% to 84% and 43% to 98%, respectively. Its sensitivity is lower for small papillary bladder tumors and carcinoma in situ. Moreover, the accuracy of WLC has been shown to be operator dependent. Given the known drawbacks of an office-based flexible cystoscopy under local anesthesia, it is recommended to perform a thorough reassessment using the rigid cystoscope under general anesthesia when patients are brought to the operating room to resect a newly diagnosed bladder tumor.

Moreover, evidence suggests that the operator’s ability to estimate the stage or grade based on the visual appearance on WLC is limited. It is also difficult to differentiate inflammatory lesions because of the previous instillation therapy from carcinoma in situ (CIS), as both will present as erythema. Although WLC remains the current standard for diagnosis and follow-up, further improvements of the technique are needed.

Fluorescence cystoscopy

Fluorescence cystoscopy or photodynamic diagnosis (PDD) can improve the visualization of BCa compared with conventional WLC and, consequently, reduced the rates of residual tumor at the first cystoscopy. PPD relies on the intravesical administration of endogenous or exogenous photosensitizing agents that cause abnormal or rapidly proliferating cells to fluoresce under a specific wavelength of light. The solution containing the photosensitizing agents is instilled in the bladder thru a transurethral catheter before surgery. By illuminating the mucosa with blue-violet light, the neoplastic cells appear red or pink against a blue background. However, false or artifact fluorescence can occur with inflammation, recent TUR, or tangential illumination of the bladder mucosa. False positives are frequent in patients who recently have had intravesical therapy, especially bacillus Calmette-Guérin.

Multiple studies have demonstrated that PDD, in addition to WLC, improves the detection rate of BCa. The tumor detection rate for WLC alone is 73% to 96% versus 90% to 96% for WLC plus PDD. The difference is even more striking for carcinoma in situ: 23% to 68% versus 91% to 97%, respectively. However, the false-positive detection rate of PDD is higher (9%–63% vs 7%–47%, respectively). A recent meta-analysis of 18 studies showed that the rate of residual tumor was significantly decreased after PDD (odds ratio [OR]: 0.28; 95% confidence interval [CI], 0.15–0.52; P <.0001), whereas the recurrence-free survival was higher at 12 and 24 months in the PDD groups relative to the WLC-only groups (log-rank = 0.00002). Several researchers have also investigated the value of PDD in the evaluation of patients with positive urine cytology in the absence of disease after standard investigations. For example, Ray and colleagues showed that BCa was detected by PDD in 32% of patients with confirmed positive urinary cytology and negative WLC. Finally, the same group also examined the role in the treatment of patients with multifocal tumors, who are at a 1.5-fold increased risk of recurrence. They showed in a series of 18 patients that the sensitivity of PDD for the detection of tumor was 97.8% compared with 69.6% for WLC.

Narrow-band imaging

For NBI, optical filters are use in the light source to narrow the bandwidth between 415 nm (blue light) and 540 nm (green light). This narrow bandwidth is strongly absorbed by hemoglobin, thus enhancing surface capillaries and blood vessels. In contrast to PDD, systems integrating WLC and NBI are already available. Since its initial report in 2008, several studies have examined the accuracy of NBI cystoscopy for the detection of BCa and showed improved detection for NBI over WLC. A meta-analysis including 8 studies (n = 1022) showed a sensitivity and specificity for NBI of 94.3% and 84.7%, respectively, versus 84.8% and 87.0% for WLC.

Optical coherence tomography

OCT is a noninvasive technique that provides cross-sectional imaging below the mucosal surface. OCT is analogous to ultrasound imaging, except that it is based on the depth detection of light rather than sound. For image acquisition, OCT is probe based and relies on WLC to identify suspicious areas that warrant further characterization. The sensitivity and specificity of OCT to detect BCa range from 84% to 100% and from 78% to 90%, respectively. OCT also has demonstrated to discriminate accurately between muscle-invasive and non–muscle-invasive tumors. Conversely, false positives may occur in the presence of scarring or inflammation of the mucosa.

Perspectives on endoscopic examination

The role of complementary approaches to endoscopic examination of the bladder remains unclear. In an era of heightened scrutiny with regard to health care costs, several investigators have hypothesized that these techniques may help in reducing the financial burden of BCa. For example, by preventing recurrences and subsequent reinterventions, PDD would potentially be cost-effective; a health technology assessment from the United Kingdom showed that management strategies involving PDD for the detection and follow-up of BCa provided some marginal financial benefits when weighing in the acquisition costs.

Transurethral resection of bladder tumors

TURBT under regional or general anesthesia is the gold standard to excise (and potentially cure) all visible tumors and to provide specimens for staging and grading of BCa. In this section, the authors discuss the preoperative and intraoperative specifics of TURBT.

Preoperative imaging

Before resection, upper tract imaging is usually recommended both to identify other sources of hematuria and to assess the upper tract urothelium because of the field change nature of urothelial carcinoma that can affect such cells in all parts of the urinary tract. Such imaging studies are routinely done as part of the workup for hematuria, the most common presentation of a bladder tumor. Indeed, the American Urologic Association’s best practices guidelines for asymptomatic hematuria suggest either intravenous urography or CT urography as the initial imaging test.

In the past decade, upper tract imaging is usually obtained with a CT urography, which allows the assessment of both the renal parenchyma and the upper tract urothelium. It is also able to rule out other genitourinary conditions that may cause hematuria, including urolithiasis, renal masses, urinary tract infection, and trauma. For these reasons, it is now the standard imaging modality for this indication and has completely replaced intravenous urography at Brigham and Women’s Hospital since 2000. Alternatively, other diagnostic modalities of the upper tract include abdominal ultrasound, intravenous urography, magnetic resonance urography, and intraoperative retrograde pyelogram. Abdominal ultrasound is sensitive in diagnosing hydronephrosis but has a limited role to identify lesions in the ureter. Magnetic resonance provides similar information compared with CT urography, yet is less accurate for calculi, takes significantly longer to perform, and costs more. However, it has the advantage of not requiring ionizing radiation. Unfortunately, there have been no direct comparisons between CT and magnetic resonance urography for the detection of urothelial carcinoma. Finally, an alternative to preoperative imaging is to perform an intraoperative retrograde pyelogram. Cowan and colleagues compared CT urography with retrograde pyelogram for the detection of urothelial carcinoma and found that the only false-negative CT urography was a small urothelial tumor that was not visible in a unopacified segment of the ureter.

Preoperative risk assessment

Basic laboratory tests should be obtained before surgery. Urine culture should be done to rule out infections, which should be treated before proceeding to this elective procedure. Complete blood count and basic metabolic profile including creatinine may provide important preoperative baseline information. A coagulation profile is often recommended and required if patients are to have the procedure under spinal anesthesia.

Preoperative risk assessment should be done meticulously, as urothelial carcinoma of the bladder is a disease of the elderly, an increasingly multi-morbid population. Patients should be assessed to ensure that they will tolerate the lithotomy position. A thorough medication history is critical. Antiplatelet agents should be discontinued 7 to 10 days before surgery when possible. In select patients, it may be necessary to continue these drugs, with an increased risk of bleeding, for medical reasons. There are data to suggest that a low dose of acetylsalicylic acid does not increase the risk of overall bleeding or reintervention. Further, another study by Carmignani and colleagues showed that TURBT under dual antithrombotic therapy (clopidogrel + acetylsalicylic acid) is feasible. However, there is a general consensus that warfarin should be discontinued at least 5 days prior and that the international normalized ratio should be checked the day of surgery.

Surgical technique

TURBT can be performed under local, spinal, or general anesthesia. The choice of technique will depend on several factors including the preoperative risk assessment as well as tumor characteristics (location, size, and anticipated depth of invasion). Local anesthesia TURBT should only be done to excise and fulgurate small/superficial tumors in select patients at increased risk of complications with regional/general anesthesia. Regional anesthesia in the form of spinal or epidural blockade is effective to treat tumors that are not on the lateral aspect of the bladder. This type of anesthesia provides the advantage of having a conscious patient who would present abdominal or shoulder-tip pain in the advent of an intraperitoneal perforation. Finally, general anesthesia with paralysis to prevent the obturator reflex is recommended for large, lateral bladder masses. An alternative to complete blockade is the obturator nerve block; however, this is seldom practiced. Antibiotics should be given in concordance with local and national practice guidelines.

Following anesthesia, patients are placed in a dorsal lithotomy position with padding of all pressure points. Special care should be taken to avoid foot drop because of neurapraxia of the common peroneal nerve, passing along the lateral proximal fibula.

The bladder should be inspected with a rigid cystoscope using both a 30° and a 70° lens. Cystoscopic findings should be redocumented and may differ from the office flexible cystoscopy, particularly in regard to the location of tumors. Thereafter, resection should be performed using a 30° lens placed through a resectoscope sheath. Irrigation must be used to facilitate visualization of the tumor, but efforts should be made to minimize overdistention and, consequently, the risk of bladder perforation. TURBT is usually performed in sterile water or glycine, as saline conducts electricity and scatters energy from monopolar cautery. Recently, bipolar resection has been introduced as an alternative approach using saline irrigation to minimize the risk of obturator reflex.

Tumors can be resected in a staged or en bloc fashion. Friable, low-grade tumors that are 3 cm or greater can be excised en bloc, often without electrical energy. The advantages of this approach for select tumors include decreased cautery artifact, avoidance of tumor fragmentation, and preservation of spatial orientation of the specimen. However, in most cases, the resection is performed in a staged-piecemeal fashion whereby each layer is carefully resected until the base of the tumor is reached. Staged resection minimizes the chance of bladder perforation. Moreover, using the cutting current is important to minimize cautery artifacts and, consequently, improve the pathologist’s ability to assess the stage and grade. In patients with suspected involvement of the muscularis propria, following complete resection, cold cup biopsies should be obtained to determine the presence of muscle invasion of the tumor base. Failure to demonstrate invasion imposes the need for repeat resection unless the decision is made to proceed to cystectomy based on factors other than muscle invasion (such as recurrent high-grade T1).

Special attention is required in cases of tumors situated near a ureteral orifice, inside a diverticulum, or in the anterior aspect of the bladder. First, cautery should be used judiciously when resection is near the ureteral orifice. In doubt, it may be cautious to leave a ureteral stent to prevent temporary obstruction from postoperative edema. Resection of diverticular tumors presents a high risk of bladder wall perforation, and accurate staging is difficult to achieve in this circumstance because the underlying detrusor is absent. For these reasons, diverticulectomy, partial cystectomy, or radical cystectomy should be considered for large or high-grade tumors arising inside a diverticulum. Regarding anterior tumors, minimal bladder filling combined with manual suprapubic compression to bring the tumor toward the resectoscope facilitates removal.

At the end of the procedure, bimanual examination of the bladder should be performed under anesthesia to accurately perform T staging. One hand is placed in the lower abdomen and 1 to 2 fingers of the other hand are placed in the anus (male) or vagina (female). Fixation (cT4) or persistence (cT3) of a palpable 3-dimensional mass after resection suggests locally advanced disease.

The current management guidelines from the National Comprehensive Cancer Network recommend repeat resection (within 6 weeks) in the following: (1) incomplete initial resection, (2) no muscle in original specimen for high-grade disease, (3) large or multifocal lesions, or (4) any T1 lesion. There is convincing evidence to support the use of repeat TURBT. For example, Herr showed that 28 out of 96 patients with non–muscle-invasive tumors were reclassified as muscle invasive at repeat resection. A prospective trial by Divrik and colleagues randomized patients with newly diagnosed T1 urothelial carcinoma of the bladder to repeat resection and adjuvant intravesical mitomycin C versus adjuvant intravesical mitomycin C without re-resection. Both the recurrence (25.7% vs 63.2%) and progression (4.1% vs 11.8%) rates were lower in patients who underwent repeat TURBT.

Imaging

Although the gold standard of BCa diagnosis is WLC cystoscopy and subsequent TURBT, many imaging techniques have emerged to better diagnose and stage the primary lesion and regional/distant spread.

Virtual cystoscopy

Virtual cystoscopy was developed in an effort to reduce discomfort and pain reported by more than one-third of patients with standard cystoscopy. Two kinds of virtual cystoscopy exist: CT virtual cystoscopy and MRI virtual cystoscopy. In a meta-analysis that pooled a total of 26 studies based on 3084 patients, the pooled sensitivities of CT virtual cystoscopy and MRI virtual cystoscopy were 94% and 91%, respectively. For the same groups, the pooled specificities were 98% and 95%, respectively. Although virtual cystoscopy may be less invasive compared with standard cystoscopy, the latter represents the gold standard recommended by existing guidelines.

Computed tomography and MRI for localized bladder cancer

According to the most recent version of the National Comprehensive Cancer Network’s guidelines for BCa, if the cystoscopic appearance of the tumor is solid, high grade, or possibly invasive, a CT scan or MRI of the abdomen or pelvis is recommended before TURBT. Previous studies have reported a sensitivity of 62% to 91% and a specificity of 63% to 95% for the detection and staging of BCa of CT scans, respectively. Studies focusing on multidetector CT showed sensitivity ranging between 89% and 91% and specificity ranging between 92% and 95%. Experts agree that CT cannot reliably differentiate non–muscle-invasive from muscle-invasive BCa. However, CT may be useful in detecting extravesical spread.

Compared with CT, MRI seems to be superior in clinical staging of BCa. On its own, staging sensitivity ranges between 68% and 80% and specificity ranges between 90% and 93%. Compared with TURBT, MRI is unlikely to be more accurate. However, TURBT is associated with a non-negligible rate of understaging (42%), whereas MRI is useful in detecting T3 to T4 disease.

Computed tomography, MRI, and PET for lymph node metastasis

The current conventional staging methods (ie, TUR pathology and cross-sectional imaging) may result in up to 25% of patients with lymph node metastases that are missed before surgery. In consequence, there is an obvious interest in the use of imaging tools to identify the presence of local and regional disease burden in the preoperative setting, as lymph node metastasis represents an important predictor of treatment success.

CT scans detect lymph node metastases in BCa with a sensitivity ranging between 31% and 50% and a specificity ranging between 68% and 100%. What has been found in direct comparisons between CT and MRI in the detection of lymph nodes preoperatively is that MRI is in general better at finding a higher number of lymph nodes and at detecting lymph nodes that are smaller (1–5 mm). Compared with CT, F-fluorodeoxyglucose PET (FDG PET) demonstrated a sensitivity of detecting metastatic disease outside of the pelvis of 54% versus 41% for CT, respectively, and comparable specificity (∼97%). In general, PET scan seems to be superior compared with CT or MRI in detecting lymph node metastases preoperatively. However, the use of PET in BCa is also limited because of the urinary excretion of FDG and subsequent poor differentiation of lesions in the bladder and adjacent lymph nodes.

Evaluating chemotherapy response using MRI

MRI is used after radiation for BCa to identify residual active malignancy. Schrier and colleagues examined 40 patients with cN1–N2 treated with chemotherapy. After patients’ second and fourth chemotherapy courses, evidence of early response was assessed with dynamic-contrast-enhanced MRI. The clinical response was determined by examining the rapidity with which the tumor and lymph nodes enhanced with contrast. The results of their study suggest that if the cancerous tissue enhanced greater than 10 seconds after arterial enhancement, those individuals could be considered responders of chemotherapy. The study reported a sensitivity of 91% and a specificity of 93%.

Perspectives on imaging

CT and MRI are considered options. Compared with TURBT, both CT and MRI may not be useful in staging purposes, unless it is to confirm or rule out extravesicle disease or metastatic spread. PET scan may be more accurate than CT and MRI at detecting lymph node metastases. The conjunctive use of standard CT with PET is not recommended. The use of MRI can serve as a radiographic biomarker in assessing the response to chemotherapy.