Prostate Cancer Screening Results from the Prostate, Lung, Colorectal, and Ovarian Cancer Randomized Screening Trial: Questions and Answers
- The Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial enrolled 154,910 men and women ages 55 to 74, to see if certain cancer screening tests help to reduce deaths from these cancers.
- Of the 76,693 men on the PLCO trial, 38,343 were randomly assigned to six annual rounds of screening with prostate-specific antigen (PSA) tests and four annual digital rectal exams (DRE) and the remaining 38,350 men were assigned to the usual care of their physicians. Annual random surveys of men in the usual-care group showed more men getting prostate cancer screening tests each year, up to 52 percent by the last year of trial screening.
- PLCO trial data showed that six rounds of annual screening for prostate cancer led to finding 22 percent more prostate cancers by seven years after the start of screening and 17 percent more prostate cancers by 10 years after the start of screening.
- Both groups had few deaths from prostate cancer, which is a reflection of good treatment practices in the U.S. and a healthy volunteer effect. The difference in the number of deaths between the groups is not statistically significant.
- What is the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial?
The Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial is a large-scale clinical trial to determine whether certain cancer screening tests can help reduce deaths from prostate, lung, colorectal, and ovarian cancer. By using screening tests for cancer, doctors may be able to discover and treat cancers that might otherwise kill a person. Many tests can identify a cancer when it is small, but only a large-scale clinical trial can prove that a test will reduce a person's chance of dying from the disease. From 1993 to 2001, 154,910 healthy men and women ages 55 to 74 enrolled in the PLCO trial at 10 centers across the United States. The PLCO trial has centers in Birmingham, Ala.; Detroit, Mich.; Denver, Colo.; Honolulu, Hawaii; Marshfield, Wisc.; Minneapolis, Minn.; Pittsburgh, Pa.; Salt Lake City, Utah (with a satellite center in Boise, Idaho); St. Louis, Mo.; and Washington, D. C.
- What tests did the PLCO participants receive?
PLCO trial participants were randomly assigned to one of two groups: the intervention group or the control group (usual-care group). In addition to annual surveys of all participants, the control group received the usual care from their health care providers, which may have included screening tests for cancers. The intervention group received:
- Prostate specific-antigen (PSA) test (men only) at the initial visit and annually for five years (6 tests)
- Digital rectal exam (men only) at initial visit and annually for three years
- Flexible sigmoidoscopy at initial visit and after three years until 1998. At that time, the interval between testing was increased to five years.
- Chest X-ray: smokers had a chest X-ray at entry and annually afterward for three years. Never-smokers had a chest X-ray at entry and annually for two years.
- CA-125 blood test (women only) at initial visit and annually for five years
- Transvaginal ultrasound (women only) at initial visit and annually for three years
- What are the results of the prostate cancer screening portion of PLCO?
Data from the PLCO trial showed that six rounds of annual screening for prostate cancer compared with community-based screening practices led to finding more prostate cancers, but did not translate into fewer prostate cancer deaths up to 10 years after the start of screening. Of the men in the screening group, on average 85 percent had PSA tests and 86 percent had digital rectal exams each round. Annual random surveys of men in the usual-care group showed more men getting prostate cancer screening tests each year, up to 52 percent by the last year of trial screening.
Seven years after the start of screening, there were 22 percent more prostate cancers diagnosed in men in the intervention group (2,820 men in the intervention group vs. 2,322 men in the usual-care group). This excess is continuing in data that has been assessed up to10 years after the start of screening, with 17 percent more cancers in men in the intervention group (3,452 men vs. 2,974 men in the usual-care group).
Both groups had few deaths from prostate cancer, which is a reflection of good treatment practices in the United States and a healthy volunteer effect. Seven years after the start of screening, there were 50 deaths from prostate cancer in the screening group and 44 deaths in the usual-care group. Ten years after the start of screening, there were 92 prostate cancer deaths in the screening group and 82 in the usual-care group. The difference in the numbers of deaths between the groups is not statistically significant.
- Did men who were screened annually have earlier-stage cancers?
The vast majority of men in both groups of the PLCO trial who developed prostate cancer were diagnosed with stage II (out of IV) disease. The number of later stage cancers was small and also similar between the two groups. However, using the Gleason scoring system, which measures how aggressive a cancer is, men in the usual-care group had more cancers that fell into the Gleason 8 to 10 range, marking them as more aggressive.
- What happened in the trial when an abnormal result was found?
Results of the PLCO trial tests were sent to the participants and to their physicians as soon as they became available. If there were any abnormal test results, the participant was referred to a physician of his choice for diagnostic follow-up tests and treatment if needed. Men in both groups who were diagnosed with similar stages of prostate cancer generally received similar treatments for their disease even though the PLCO trial study design did not mandate specific therapies.
- What is happening to the men on the PLCO trial?
Men taking part in the study were informed about these findings in mid-March 2009. Screening tests in the PLCO trial ended in 2006. The men will continue to be followed by study staff for at least 13 years from their entry onto the trial to track for the development of prostate cancer and other trial endpoints of lung and colorectal cancer. They can speak to their own physicians about whether or not to continue to be screened for prostate cancer.
- What did results from the European study of prostate cancer screening, released at the same time as the PLCO prostate results, show?
The European Randomized Study of Screening for Prostate Cancer (ERSPC) is a study of 182,000 European men ages 50 to 74, half of whom are screened in most centers every four years with a PSA test. The study found that after a median follow-up of nine years, there were 20 percent fewer deaths from prostate cancer in the screened men than in the men in the control group in the age group 55 to 69. There were over 40 percent more prostate cancers diagnosed in the men in the screened group than in the men in the control group. The investigators calculate that 1,410 men would have to be screened and 48 additional cases of prostate cancer would need to be treated to prevent just one death from prostate cancer.
- Why do the European results differ from the PLCO trial results?
Importantly, the study results of the PLCO and ERSPC are identical at seven years. The benefits from ERSPC are seen later, especially after 10 years. PLCO has not reported data yet at those time points. However, there are many differences between the study designs of the two trials, all of which may add to the different results. Among these differences are:
- The PLCO trial screened men on an annual basis and ERSPC screened, typically, every four years. The PLCO trial reported the results for all six rounds of screening while ERSPC reported on the first round and later rounds when available.
- The PLCO trial used a cut-off value of higher than 4.0 ng/mL for a "positive" PSA result while most ERSPC centers used 3.0 ng/mL or higher.
- The men in the ERSPC were chosen from predominately white populations in The Netherlands, Belgium, Sweden, Finland, Italy, Spain and Switzerland. The PLCO trial enrolled a population that was approximately 15 percent minority, chosen to best reflect the U.S. population.
- The PLCO trial included DREs for all men as part of the screening while not all the ERSPC sites included DREs.
- Because of the differences in the way medicine is practiced in Europe vs. the United States, it is presumed that the men in the ERSPC control group were not screened to as large a degree as the men in the PLCO trial control (usual-care) group. In the PLCO trial, each year a random sample of men in the usual-care group received a survey asking about their screening practices. In the sixth year of the trial, an estimated 52 percent of the men in the usual-care group received a PSA within that year.
The two trials provide important information on the utility of prostate cancer screening but it is difficult to compare them directly.
- Does NCI recommend screening for prostate cancer? Will that recommendation change?
NCI does not have a recommendation about prostate cancer screening. The Agency for Health Care Quality and Research, the federal agency tasked with making screening recommendations, convenes the U.S. Preventive Services Task Force, an independent panel of experts in primary care and prevention that systematically reviews the evidence of effectiveness and develops recommendations for clinical preventive services. This task force recently concluded that there is insufficient evidence to assess the balance of benefits and harms of prostate cancer screening in men younger than age 75 and recommended against prostate cancer screening in men age 75 and older. These new trial results from the PLCO and ERSPC may be used in future deliberations about prostate cancer screening by this group.
NCI reports levels of evidence about prostate cancer screening as part of its Physician's Data Query (PDQ) site at http://www.cancer.gov/cancertopics/pdq/screening/prostate/healthprofessional. This information was last updated in November 2008 and a new update will be considered based on the PLCO and ERSPC findings.
- What are the average costs of the most common prostate cancer treatments?
From 1995 through 2004, the overall costs of treating all cancers (including prostate) increased by an estimated 75 percent, due in part to advances in diagnostic and surgical techniques and pharmaceutical innovations. Cancer treatment cost an estimated $72.1 billion in 2004, just under five percent of total spending for medical treatment in the United States. Much of the cost of treating cancer has been and will be borne by Medicare because patients 65 years of age and older accounted for 56.2 percent of all cancer diagnoses in the United States between 1998 and 2002.
Based on Medicare costs for men age 65 and older who were diagnosed with prostate cancer between 1991 and 2002, from two months before diagnosis to 12 months after diagnosis, Medicare paid an average of $18,261 for the typical man's treatment. The average payment for prostate cancer declined from 1991 through 2002. This decrease can be explained by a 29 percent drop in cancer-related surgeries. The reduction in the use of radical prostatectomy, however, was offset by a 14 percent increase in the use of radiation therapy from 1991 to 2002. The mean annual costs of prostate cancer-related surgery during this period ranged from $3,451 to $7,987 higher than radiation therapy costs. Although the costs associated with prostate cancer-related surgery are greater than for radiation therapy, the mean cost of prostate cancer surgery has been declining while radiation costs have risen.
About prostate cancer screening tests
- What is the prostate-specific antigen (PSA) test?
PSA is a protein produced by the cells of the prostate gland. The PSA test measures the level of PSA in the blood. The doctor takes a blood sample, and the amount of PSA is measured in a laboratory. Because PSA is produced by the body and can be used to detect disease, it is sometimes called a biological marker or tumor marker.
It is normal for men to have low levels of PSA in their blood; however, prostate cancer or benign (not cancerous) conditions can increase PSA levels. As men age, both benign prostate conditions and prostate cancer become more common. Therefore, PSA levels alone do not give doctors enough information to distinguish between benign prostate conditions and cancer. However, a doctor will take the result of the PSA test into account when deciding whether to check further for signs of prostate cancer.
- How are PSA test results reported?
PSA test results report the level of PSA detected in the blood. The test results are usually reported as nanograms of PSA per milliliter (ng/mL) of blood. Unlike some other laboratory tests, there is no specific "normal" or "abnormal" PSA level, and reference ranges can vary slightly between laboratories. The PSA test cannot determine whether a man has prostate cancer or a noncancerous condition. Various medical factors (such as an infection) can cause PSA levels to fluctuate and a man's PSA level tends to increase with age so the results of one PSA test do not necessarily indicate a need for a biopsy. In addition, some studies have shown that African American men are more likely to have elevated PSA levels and not have cancer than white men.
Doctors have different opinions about what PSA level should prompt a biopsy to look for prostate cancer. In the past, most doctors considered PSA levels below 4.0 ng/mL as "normal." Others considered a PSA of 2.5 ng/mL or 3.0 ng/mL to be the upper limit of normal. However, results from the NCI-sponsored Prostate Cancer Prevention Trial found prostate cancer in about 15 percent of men with PSA levels below 4.0 ng/mL, indicating that there really is no such thing as a "normal" PSA level. In addition, even PSA levels above 4 ng/mL are more likely to be due to noncancerous conditions than to prostate cancer and, in fact, only about 25 percent to 30 percent of men with PSA levels between 4 and 10 ng/mL will be found to have prostate cancer on biopsy.
In general, the higher a man's PSA level, the more likely it is that cancer is present. PSA levels higher than 10.0 ng/mL are associated with prostate cancer in more than 50 percent of men and levels of 20.0 ng/mL or higher are more likely to be associated with metastatic prostate cancer.
Scientists are looking for a blood or urine test that will distinguish between cancerous and benign conditions, and between slow-growing cancers and fast-growing, potentially lethal cancers. Some doctors continue to use the 4.0 ng/mL measure as the point at which to recommend a prostate biopsy, while others use a lower level. Some doctors also compare changes in PSA levels over time (PSA velocity or PSA doubling time) or adjust PSA levels based on a man's age (see Question 17).
- What is a digital rectal exam?
A digital rectal exam, or DRE, is a physical exam in which a health professional feels for abnormalities in a man's prostate gland. Because the prostate is located near the rectum, a clinician can feel it by inserting a gloved finger into the rectum. A cancer can often be felt as a hard lump. A DRE exam is reported as either normal or abnormal, and the test is limited by how much of the prostate a clinician is able to feel.
- What if screening test results show an elevated PSA level or an abnormal DRE?
A man should discuss all test results with his doctor. There can be different reasons for an elevated PSA level, including prostate cancer, benign prostate enlargement, inflammation, infection, age, and race.
If no other symptoms suggest cancer, the doctor may recommend repeating DRE and PSA tests regularly to watch for any changes. If a man's PSA levels have been increasing or if a suspicious lump is detected during the DRE, the doctor may recommend other tests to determine if there is cancer or another problem in the prostate. If cancer is suspected, a biopsy is needed to determine if cancer is present in the prostate. During a biopsy, samples of prostate tissue are removed, usually with a needle, and viewed under a microscope. The doctor may use ultrasound to view the prostate during the biopsy, but ultrasound cannot be used alone to tell if cancer is present
- What are some of the limitations of the PSA test?
- Detection does not always mean saving lives: When used in screening, the PSA test can detect small tumors. However, finding a small tumor does not necessarily reduce a man's chance of dying from prostate cancer. PSA testing may identify very slow-growing tumors that are unlikely to threaten a man's life. Also, PSA testing may not help a man with a fast-growing or aggressive cancer that has already spread to other parts of his body before being detected.
- False-positive tests: False-positive test results (also called false positives) occur when the PSA level is elevated but no cancer is actually present. False positives may lead to additional medical procedures that have potential risks and significant financial costs and can create anxietyfor the patient and his family. Most men with an elevated PSA test turn out not to have cancer; only 25 to 30 percent of men who have a biopsy due to elevated PSA levels actually have prostate cancer.
- False-negative tests: False-negative test results (also called false negatives) occur when the PSA level is in the normal range even though prostate cancer is actually present. Most prostate cancers are slow-growing and may exist for decades before they are large enough to cause symptoms. Subsequent PSA tests may be elevated and indicate a problem.
- What research is being done to improve the PSA test?
Scientists are researching new ways to distinguish between cancerous and benign prostate conditions, and between slow-growing cancers and fast-growing, potentially lethal cancers. Some of the methods being studied are:
- PSA velocity: PSA velocity is the change in PSA levels over time. A sharp rise in the PSA level raises the suspicion of cancer and may indicate a fast growing cancer. More studies are needed to determine if high PSA velocity more accurately detects prostate cancer than just a single PSA level.
- Age-adjusted PSA: Age is an important factor in increasing PSA levels. For this reason, some doctors use age-adjusted PSA levels to determine when diagnostic tests are needed. When age-adjusted PSA levels are used, a different PSA level is defined as normal for each 10-year age group. Doctors who use this method generally suggest that men younger than age 50 should have a PSA level below 2.4 ng/mL, while a PSA level up to 6.5 ng/mL would be considered normal for men in their seventies. Doctors do not agree about the accuracy and usefulness of age-adjusted PSA levels.
- PSA density: PSA density considers the relationship of the PSA level to the size of the prostate. In other words, an elevated PSA might not arouse suspicion if a man has a very enlarged prostate. The use of PSA density to interpret PSA results is controversial because cancer might be overlooked in a man with an enlarged prostate.
- Free versus attached PSA: PSA circulates in the blood in two forms: free (at least three inactive forms of PSA) or attached to a protein molecule. The free PSA test is more often used for men who have higher PSA values. Free PSA may help tell what kind of prostate problem a man has. With benign prostate conditions, there is more free PSA, while cancer produces more of the attached form. If a man's attached PSA is high but his free PSA is not, the presence of cancer is more likely.
- ProPSA vs. Total Free PSA: One kind of free PSA is the precursor form of PSA called proPSA, which has several different types. A measure of the amount of one of these types known as [-2]proPSA, when compared to overall free PSA, is a measurement called %[-2]proPSA. This measure seems to be very useful in determining if prostate cancer is present in men with PSA levels up to 10 ng/mL and is under study in NCI's Early Detection Research Network.
- Alteration of PSA cutoff level: Some researchers have suggested lowering the cut-off levels that determine if a PSA measurement is normal or elevated. For example, a number of studies have used cut-off levels of 2.5 or 3.0 ng/mL (rather than 4.0 ng/mL). In such studies, PSA measurements above 2.5 or 3.0 ng/mL are considered elevated. Researchers hope that using these lower cutoff levels will increase the chance of detecting prostate cancer; however, this method may also increase overdiagnosis and false-positive test results and lead to unnecessary medical procedures.
- What other methods are being studied to detect prostate cancer?
NCI is supporting researchers nationwide who are studying ways to better detect prostate cancer and to detect which prostate cancers will be aggressive. Ongoing research to find new early detection tests include:
- Micro-RNA based assays: Micro-RNA is a tiny piece of ribonucleic acid (RNA) that binds to matching pieces of messenger RNA (mRNA) to make it double-stranded. This binding may cause a decrease in the production of the corresponding protein. A profile of mRNA expression is being studied to determine if it adds information that would be helpful in determining if men with high PSA levels, but no abnormal results on a digital rectal exam, have prostate cancer.
- Methylation of certain genes: Methylation of DNA occurs frequently at specific areas in the DNA strand near the promoters of the gene. These areas are known as CpG islands. Modifications in CpG islands often inactivate tumor suppressor genes' expression. The GSTPi 1 gene is methylated in prostate cancer and some precancerous prostate conditions, and may be a marker of the transition from a normal prostate cell to one primed for development of cancer.
- Gene fusion: The joining of DNA from different parts of the genome may be an important event in the development of prostate cancers. Members of the ETS family of oncogenes (cancer-causing genes) are frequently fused to other genes. About half of prostate cancers may have this change, making a test for such fusions a potential detection test.
- Detection of differential metabolites: Molecules produced by the body's metabolic processes may be able to differentiate between benign prostate tissue vs. localized and metastatic prostate cancer. One such molecule, known as sarcosine, may be associated with prostate cancer invasiveness and aggressiveness. Research is ongoing to see if sarcosine can be used as a test.
- PCA3: PCA3, or DD3, is a prostate-specific mRNA reported to be frequently overexpressed in prostate tumor cells. A urine test for this RNA, used in addition to current prostate cancer screening tests, has the potential to improve detection and is under study.
- Proteo-imaging: Proteo-imaging is the ability to localize and follow changes at the molecular level by imaging the protein distributions in specific tissues. Being able to see different patterns of protein expression in healthy prostate tissue versus precancerous conditions may help classify early prostate changes that may one day lead to cancer.
About the PLCO trial
- What is known from the PLCO trial about the value of screening tests for lung, colorectal, and ovarian cancers?
No mortality data from lung, colorectal, or ovarian cancers has been reported as yet, but results of the initial round of screening for lung, colorectal and ovarian cancers are known:
- Lung: A total of 77,465 participants were randomly assigned to the intervention arm and had a chest X-ray when joining the trial. Of these, 5,991 X-rays were suspicious for lung cancer (8.9 percent overall with 8.2 percent of the men vs. 9.6 percent for the women). Smokers and older participants were more likely to have a suspicious test result. Of the 5,991 participants with suspicious X-rays, 206 had a biopsy within 12 months of the X-ray, of which 126 had lung cancer. Overall, 1.9 lung cancers were found for each 1,000 chest X-rays done. For current smokers, the rate was 6.3 per 1,000 and for former smokers who had quit within 15 years of study enrollment, the rate was 4.9 per 1,000. Almost 44 percent of all lung cancers found were stage I (out of IV).
- Colorectal: A total of 64,658 participants had a screening flexible sigmoidoscopy upon entering the trial, and at least one polyp or mass was identified in 23.4 percent of them (15,150). Of these, nearly 75 percent (11,241) had follow-up endoscopic procedures and nearly half had either colorectal cancer or an adenoma. Cancer was diagnosed in 1.6 percent, advanced adenomas in 19.1 percent, and non-advanced adenomas in 31.4 percent of those screened. For every thousand people screened, 2.4 had cancer, 27.8 had an advanced adenoma, and 45.6 had a non-advanced adenoma.
- Ovary: Of the 39, 115 women randomized to receive screening for ovarian cancer, 28,816 had at least one test: either a transvaginal ultrasound (TVU) or a CA-125 blood test. An abnormal TVU was found in 1,228 (4.7 percent) and an abnormal CA-125 in 402 (1.4 percent) of those screened. Twenty-nine cancers were found, nine of which had a low potential for malignancy. Ovarian cancers were found in both early and late stages.
- What other research is supported by the PLCO trial?
In addition to the PLCO trial being a randomized, controlled clinical trial of more than 150,000 people to evaluate if screening for these major cancers reduces cancer deaths, it is a prospective, epidemiologic cohort study looking at cancer risk factors, dietary questions, rates of all cancers, and is an invaluable source for a biorepository of almost three million specimens.
- What kind of information is collected for epidemiologic studies?
All PLCO trial participants provided information about their health and lifestyle over the 17 years of the trial to date. This information included:
- The Baseline Questionnaire, completed at the time of enrollment, collected information on demographics, personal and family history, lifestyle habits (including smoking) and history of screening for prostate, lung, colorectal and ovarian cancer.
- The Annual Study Update questionnaire is mailed to all participants yearly for at least 13 years to identify all cancers with a particular emphasis on cancers of the prostate, lung, colorectum, and ovary, as well as all deaths that occur among both screened and control subjects during the trial.
- Dietary questionnaires to look at relationships between diet and cancer were given to all participants. Participants in the screened arm were asked to complete two full-length questionnaires while those in the control arm were asked to complete one questionnaire.
- A risk factor questionnaire was mailed to all participants in 2006. Risk factors can include habits such as smoking, drinking alcohol, diet, and other activities that may put a person at risk for disease.
- What is in the PLCO Biorepository? Why is it important?
The PLCO Biorepository has nearly three million biologic specimens that were collected during the six annual trial screens. It has several distinct advantages over other biorepositories, including the fact that the specimens are collected at each screen so that serial samples are available. Additionally, these samples are collected prospectively before a cancer is diagnosed. There is also detailed background and clinical data associated with the specimens, and the large sample size provides statistical power for researchers.
Data and specimens currently available for etiologic and early marker research include: baseline demographic data and risk factor information on participants, food frequency questionnaires on all participants, information on all-cancer incidence and selected other medical conditions, fractionated blood specimens collected annually up to six times from screening arm participants, cryopreserved whole blood from screening arm participants, and buccal cell samples (cells from the lining of the mouth) from control arm participants. In addition, the PLCO Pathology Specimen Collection has pathology tissue from PLCO trial participants with cancer and constructs tissue microarrays (slides with large numbers of tissues to streamline testing) from these samples. Prostate cancer, colorectal cancer, colorectal adenomas, and ovarian cancers have been processed and other cancers may be added.
- What kind of information has been discovered using the PLCO Biorepository?
- The PLCO trial is contributing samples for breast cancer and prostate cancer studies for NCI's Cancer Genetic Markers of Susceptibility (CGEMS) project. In 2007, CGEMS researchers reported that a variation in a portion of DNA strongly predicts prostate cancer risk and that this common variation may be responsible for up to 20 percent of prostate cancer cases in white men in the United States. This gene variation was discovered on chromosome 8 and is marked by a number of single nucleotide polymorphisms (SNPs), including rs6983267.
- Five institutions are validating their most promising ovarian cancer markers using high-quality blood samples from the PLCO trial, including some pre-diagnostic samples from women who developed cancer during the trial. Coordinated by NCI's Early Detection Research Network, the study could show whether panels of markers can detect ovarian cancer in blood collected six months or more before cancer was discovered. The markers under study might improve upon the sensitivity of the protein CA-125 test. Results are expected later in 2009.
- NCI researchers have shown that abnormal white blood cells can be present in patients' blood more than six years prior to the diagnosis of a chronic form of lymphocytic leukemia (CLL). This finding may lead to a better understanding of the cellular changes that characterize the earliest stages of the disease and how it progresses. The research team identified 45 individuals, among the more than 77,000 participants in the PLCO trial, who were cancer-free upon entering the trial and were enrolled in the screened arm. These individuals had frozen blood samples available for analysis that had been collected upon their enrollment in the PLCO trial, and later were diagnosed with CLL.
- How do scientists gain use of the PLCO Biorepository?
The PLCO Biorepository Resource is available to all qualified researchers through the Etiologic and Early Market Studies program (EEMS). EEMS accepts proposals for access twice a year. For more information, visit the PLCO Program Administrative Resource website at http://www.parplco.org.
For a press release on the PLCO prostate cancer results, please go to http://www.cancer.gov/newscenter/pressreleases/PLCOprostateResults.