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Qualit y control The need for a lower total PSA cut-off value with PSA assays calibrated to the new WHO standard The recalibration of PSA assays to the new World Health Organisation WHO 96/670 standard has certainly led to improved harmonisation of testing, but it has not yet resulted in the complete conformity expected by many clinicians and laboratory managers. There is also an urgent need for manufacturers to redefine a valid clinical decision limit after PSA assay recalibration to WHO standard, in order to maintain a similar clinical performance of the measurement. The consequence of not providing a new cut-off value is that a higher number of PSA results may be interpreted as negative in the presence of prostate cancer. by Dr V. Jarrige Prostate cancer is the second leading cause of cancer death in men, exceeded only by lung cancer. It is the most common form of malignancy in Europeans, with its incidence increasing more than with any other cancer over the last 20 years. According to WHO, about 190,000 new cases occur each year. Prostatespecific antigen (PSA) is the serum biomarker most widely used for early detection of prostate cancer and monitoring of patients with the disease. As men age, PSA levels generally rise. The most frequent reasons for elevated serum PSA values are benign prostatic hyperplasia (BPH) or prostate cancer. Measurements of total PSA allow detection of cancer as early as five years before symptoms appear [1]. Clinicians have always been aware that PSA assays may differ significantly from one manufacturer to another; the drive towards recalibration was designed to address this issue. However, recalibration alone has no impact on the improvement in the clinical interpretation of results and may have led to the erroneous assumption that all assays are now the same. More European countries now request laboratories to use PSA assays that are calibrated to the WHO 96/670 standard. However, this standard is not the one that was used to establish the clinically relevant PSA cut-off value of 4.0 ng/mL. (This was defined using the original Hybritech Tandem-R PSA calibration system). Despite this, the cut-off value of 4.0 ng/mL is still used with the WHO-standardised assay from many manufacturers. Different results can still be produced by different PSA assays even if they are standardised to the same WHO standard; this is because variations in assay architecture and antibody choice prevent full achievement of standardisation. The use of the WHO standard thus only improves harmonisation between assays [2]. The use of the 4.0 ng/mL cut-off with results Figure 1. Impact of different PSA standardisation interpretation of results and cut-off definitions. generated by WHO calibrated assays can affect the clinical sensitivity and specificity of the assay, meaning that the results need to be interpreted differently [Figure 1]. Despite the still-occurring differences in results from different WHO-calibrated assays for either PSA or free PSA, it is nevertheless highly desirable for these assays to be traceable to WHO reference preparations. More and more companies are developing solutions to that end. As an example, Beckman Coulter has decided to develop a second calibration protocol for its Access Hybritech PSA and Access Hybritech free PSA assays that allows traceability to WHO reference preparations while retaining the calibration to the original Hybritech Tandem-R PSA and Hybritech Tandem-R free PSA assays. Beckman Coulter have redefined the cut-off value for their new WHO 96/670 recalibrated Access Hybritech PSA assay to 3.1 ng/mL, but remind users that this is not automatically applicable to other methods. Hybritech - the first gold standard for PSA testing In 1986, the Hybritech Tandem-R assay became the first PSA assay to be approved for prostate cancer monitoring by the Food and Drug Administration (FDA) in the US. This set the benchmark for all PSA assays that followed. In 1994, 4.0 ng/mL was identified as the most appropriate clinical decision point for the detection of prostate cancer for the Hybritech PSA assay [3]. The original Hybritech Tandem-R calibration was based on an internal reference preparation of purified human PSA, and the clinical PSA cut-off was established as 4.0 ng/mL on the basis of results of samples from over 6,600 men who were tested by the assay calibrated with the Hybritech calibration system [4]. The original Hybritech Tandem-R assay is now available on Beckman Coulter Access immunoassay systems. The assay is used in conjunction with digital rectal examination (DRE) to aid in the detection of prostate cancer in men aged 50 years or older, to assess their prognosis and to monitor the effectiveness of any treatment. When used in conjunction with Hybritech free PSA assay, it also helps to differentiate between prostate cancer and benign conditions. The efficacy of early prostate cancer detection using PSA testing is currently the purpose of the 10-year European Randomised Study for Screening of Prostate Cancer Figure 2. Equimolar recognition of complexed and free PSA forms in serum samples is essential for accurate PSA testing: a non-equimolar assay may increase both false positive and false negative results. (ERSPC), which is expected to report its findings between 2008 and 2010. Hybritech PSA is the method of choice for the ERSPC study. The need for equimolarity in PSA testing There were significant variations in PSA results among the early non-equimolar PSA assays. Equimolar recognition of free and complexed PSA forms is essential for accurate PSA testing. Inaccurate quantification of PSA around the 4.0 ng/mL cut-off can yield a false positive or a false negative result, depending on the direction of the distortion, and may thus lead to inappropriate management of the patient [Figure 2]. In order to compensate for the non-equimolar response of some PSA assays, Thomas Stanley, a clinical urologist at Stanford University, proposed in the mid-1990s a PSA standard containing both complexed and free PSA in a ratio of 90:10 respectively [5]. This became the basis for the new standard adopted by WHO in 1999, which has its mass assigned using a method based on the use of a particular molar extinction coefficient, different from the original Hybritech standard. Although the original intention was to establish an ‘equimolarity standard’, this has actually led to the creation of a new WHO ‘mass standard’ for PSA. However, many clinicians are still unaware that restandardising a PSA assay from an original Hybritech calibration to a WHO calibration may result in potential under-recovery of PSA values. This is because assays calibrated to the new WHO standard show a negative bias in mass units compared with the Hybritech calibrated assays [6]. The original Hybritech Tandem-R calibration was based on an internal reference preparation of purified human PSA and yields PSA results about 20% higher than the new WHO standard [6]. The European Group As published in April 2007 on Tumour Markers (EGTM) clearly recommends that "every laboratory report should contain the name of the assay used and a valid reference range, specifically generated for this assay." In addition, some European countries require laboratories to report PSA and free PSA values standardised to WHO 96/670 and 96/668, respectively. cut-off [2]. During this process it became apparent that the different calibrations yielded different results, indicating that the 4.0 ng/mL cut-off would not provide optimal clinical sensitivity and specificity for the WHO calibration. The 3.1 ng/mL cut-off for the WHO calibrated method was compared against the original Hybritech data set that was used to determine the cut of 4.0 ng/mL. From Recalibrating for WHO this, the new cut-off of 3.1 ng/mL was standards verified as being appropriate [Table 1]. In order to verify the Hybritech standard A sensitivity and specificity of 81.6% and against the WHO standard, Beckman 48.0% respectively was maintained at this Coulter Immunodiagnostics (BCI) cut-off [4]. developed WHO primary calibrators to Use of the new cut-off of 3.1 ng/mL determine the ‘offset’ when compared does not impact the clinical sensitivity of tests involving the ratio of free PSA to total PSA (percentage of free PSA) [Table 2] because the WHO calibration values for both Table 1. WHO calibration 3.1 ng/mL total PSA cut-off is clinically total and free PSA equivalent to Hybritech calibration 4.0 ng/mL cut-off. exhibit similar directly against the original Hybritech differences (approximately 20%) from calibration. This indicated how much the Hybritech calibration values. adjustment was needed for both total and free PSA across the range of the assay. Why is a lower PSA cut-off imporWhen Beckman Coulter carried this out tant after WHO it was found an adjustment of 20% was recalibration? needed for both assays. When samples taken from men with The validation process involved a statis prostate cancer were evaluated using tical analysis of how the WHO standardi- a 3.1 or a 4.0 ng/mL cut-off for the sation impacts on the original Hybritech WHO calibration, use of the higher data used to establish the 4.0 ng/mL cut-off missed 15% of prostate cancers which were detected by using the cutoff of 3.1 ng/mL [Table 3]. Table 3a shows PSA results for cancer patients using a 4.0 ng/ mL cut-off with the Hybritech calibration and a 3.1 ng/mL cut-off with the Table 2. Equivalent percentage free PSA results for the Hybritech and WHO calibraWHO calibrations. tion. Table 3b AE and Schroder FH. On the standardisation of total prostate-specific antigen: an exercise with two reference preparations. Clin Chem Lab Med 1999; 37: 545-552. 3. Catalona WJ et al. Selection Table 3. Distribution, by PSA cut-off, of subjects tested for prostate of optimal PSA cancer by PSA test. cutoff for early shows the PSA results for the same can- detection of prostate cancer, ROC cer patients when their samples were curves. J Urol 1994; 152:2037–2042. analysed using a 4.0 ng/mL cut-off with 4. Catalona WJ et al. Comparison of both the Hybritech and the WHO cali- digital rectal examination and serum brations. It is apparent that using prostate specific antigen in the early the 4.0 ng/mL cut-off with the detection of prostate cancer: Results WHO calibration fails to pick up of a multicenter clinical trial of 6,630 38 of the 255 patients (15%) whose men. J Urol 1994; 151: 1283–1290. prostate cancer may be missed as a 5. Stamey TA, Teplow DB, Graves result. Laboratories need to make two HC. Identity of PSA purified from key points clear to their clinicians. seminal fluid by different methods: First, there is a new cut-off value of 3.1 comparison by amino acid analysis ng/mL for the WHO calibrated Access and assigned extinction coefficients. Hybritech PSA assay. Secondly, this Prostate 1995; 27: 198–203. new cutoff cannot be automatically 6. Link RE et al. Variation in prosapplied to other methods, as cut- tate specific antigen results from 2 off validation must be assay specific different assay platforms: Clinical according to EGTM. It is important impact on 2,304 patients undergothat other manufacturers invest in the ing prostate cancer screening. J Urol individual validation and verification 2004; 171: 2234–2238. of their WHO calibrated assays. 7. Semjonow A, Albrecht W, Bialk P, Gerl A, Lamerz R, Schmid HP and van Further information Poppel H: Tumour markers in prosTechnical information for Beckman Coulter assays tate cancer: EGTM recommendations. on the Hybritech calibration and WHO calibration Anticancer Res 1999; 19: 2799-2801. options, the lower total PSA cut-off required when calibrating to the WHO standard and QC values for Hybritech and WHO calibrations are available from [email protected]. Please note that the ������ WHO calibrated assay is not currently available in the US. References 1. H. Ballentine Carter et al. Longitudinal Evaluation of ProstateSpecific Antigen Levels in Men With and Without Prostate Disease. JAMA 1992; 267: No 16. 2. Blijenberg BG, Storm BN, Kruger The author Veronique Jarrige, Ph.D. European Scientific Manager, Immunodiagnostics, Beckman Coulter Europe, c/o 22 rue Juste-Olivier, P.O. Box 1044, 1260 Nyon 1, Switzerland Tel +33 490 50 64 72 e-mail: [email protected] Go to Hotline www.cli-online.com & tick 23465