Streamlined Multi-Attribute Assessment of an Array of Clinical-Stage Antibodies: Relationship Between Degradation and Stability.

Clinical antibodies are an important class of drugs for the treatment of both chronic and acute diseases. Their manufacturability is subject to evaluation to ensure product quality and efficacy. One critical quality attribute is deamidation, a non-enzymatic process that is observed to occur during thermal stress, at low or high pH, or a combination thereof. Deamidation may induce antibody instability and lead to aggregation, which may pose immunogenicity concerns. The introduction of a negative charge via deamidation may impact the desired therapeutic function (i) within the complementarity-determining region, potentially causing loss of efficacy; or (ii) within the fragment crystallizable region, limiting the effector function involving antibody-dependent cellular cytotoxicity. Here we describe a transformative solution that allows for a comparative assessment of deamidation and its impact on stability and aggregation. The innovative streamlined method evaluates the intact protein in its formulation conditions. This breakthrough platform technology is comprised of a quantum cascade laser microscope, a slide cell array that allows for flexibility in the design of experiments, and dedicated software. The enhanced spectral resolution is achieved using two-dimensional correlation, co-distribution, and two-trace two-dimensional correlation spectroscopies that reveal the molecular impact of deamidation. Eight re-engineered immunoglobulin G4 scaffold clinical antibodies under control and forced degradation conditions were evaluated for deamidation and aggregation. We determined the site of deamidation, the overall extent of deamidation, and where applicable, whether the deamidation event led to self-association or aggregation of the clinical antibody and the molecular events that led to the instability. The results were confirmed using orthogonal techniques for four of the samples.

BPSA: A Novel Serum Marker for Benign Prostatic Hyperplasia.

Free prostate-specific antigen (fPSA) testing was developed and approved for widespread use despite the lack of knowledge regarding the underlying biologic basis for its ability to discriminate between benign prostatic hyperplasia (BPH) and prostate cancer. We hypothesized that the relationship of total PSA to prostate volume was due primarily to the fPSA component of serum PSA, and we studied the molecular forms of PSA found in prostate tissue. Later, more sophisticated studies resulted in the discovery of BPSA (benign PSA), a novel form of fPSA associated with nodular hyperplasia of the transition zone (TZ). We found that the serum BPSA level is highly correlated with TZ and total prostate volume. In our most recent studies, we found that BPSA correlates better with TZ volume than does PSA and can predict clinically significant prostate enlargement better than PSA or fPSA. Furthermore, the relation of BPSA and fPSA to total prostate and TZ volumes is independent of age.

Effects of systematic 12-core biopsy on the performance of percent free prostate specific antigen for prostate cancer detection.

PURPOSE: The performance characteristics of percent free (f) prostate specific antigen (PSA) for differentiating between benign prostatic hyperplasia and prostate cancer were originally established using primarily sextant biopsy. We determined whether the addition of 6 laterally directed cores to the traditional sextant prostate biopsy affects the performance of percent fPSA. MATERIALS AND METHODS: We retrospectively evaluated a cohort of 350 consecutive biopsies in men with negative digital rectal examinations and PSA between 4 and 10 ng/ml who underwent systematic 12 core biopsy (S12C) biopsy at Scott Department of Urology between March 1999 and January 2003. The effects of 6 additional, laterally directed biopsies on the sensitivity, specificity and area under the ROC curve for percent fPSA was evaluated in the 277 men in whom percent fPSA was measured. RESULTS: Cancers detected exclusively in the 6 laterally directed cores were associated with percent fPSA values similar to those in patients with a benign S12C biopsy. This resulted in a modest and yet predictable decrease in the sensitivity of percent fPSA at each biopsy threshold value without affecting specificity. There was a nonstatistically significant decrease in the area under the ROC curve with the addition of 6 laterally directed cores to sextant biopsy (medial sextant cores 0.66 vs S12C 0.60). CONCLUSIONS: The 12 core biopsy strategies have a higher cancer detection rate than sextant biopsies and they are gaining widespread acceptance. The addition of 6 laterally directed cores to traditional sextant biopsy may result in a modest decrease in the sensitivity of percent fPSA at each selected biopsy threshold without affecting specificity.

Impact of unilateral interposition sural nerve grafting on recovery of urinary function after radical prostatectomy.

OBJECTIVES: To test the hypothesis that unilateral sural nerve graft (SNG) interposition may improve the rate of urinary function (UF) recovery after radical retropubic prostatectomy (RRP) in patients undergoing unilateral nerve resection (UNR). METHODS: We studied 111 consecutive patients who underwent RRP with purposeful UNR performed by a single surgeon. Of the 111 patients, 53 underwent unilateral SNG interposition. All patients were invited to complete a questionnaire that included the validated University of California, Los Angeles, Prostate Cancer Index. The time to UF recovery above the median value of the group and urinary control status were evaluated. RESULTS: The median follow-up was 26 and 12 months for the UNR and UNR+SNG patients, respectively. At 12 months after RRP, 94.7% of patients with UNR+SNG reported having complete urinary control or leakage of only a few drops of urine compared with 58.3% of patients with UNR alone (P = 0.012). In multivariate Cox regression models, UNR+SNG was associated with a 9.95 times greater rate of reaching a UF score above the median versus UNR alone (P <0.001). In multivariate logistic regression analyses, SNG status increased the odds of having complete urinary control or leakage of only a few drops of urine by 14.99 and 29.19 at 6 and 12 months after RRP, respectively (both P <0.05). CONCLUSIONS: In patients undergoing UNR surgery, SNG interposition is associated with a greater rate of UF recovery and a higher likelihood of urinary control after RRP. These findings need to be validated in larger, multicenter, prospective, randomized studies.

Serum BPSA outperforms both total PSA and free PSA as a predictor of prostatic enlargement in men without prostate cancer.

OBJECTIVES: To determine whether the serum concentration of BPSA, a distinct form of free prostate-specific antigen (PSA) enriched in the nodular transition zone (TZ) tissue of benign prostatic hyperplasia (BPH), can predict TZ volume and diagnose BPH-associated prostatic enlargement in patients without prostate cancer. METHODS: We studied 91 consecutive patients without prostate cancer who underwent a 10-core or greater biopsy of the prostate. The associations between prostate volume, age, International Prostate Symptom Score, and serum concentrations of PSA, free PSA, and BPSA were evaluated by receiver operating characteristic curve and linear and binary logistic regression analyses. RESULTS: BPSA and free PSA showed stronger correlations with both age (BPSA = 0.38, free PSA = 0.40, PSA = 0.24) and TZ volume (BPSA = 0.67, free PSA = 0.64, PSA = 0.55) than did PSA. The percent free PSA had no statistically significant correlation with TZ volume (P = 0.08). Subtraction of BPSA from free PSA reduced its correlation with TZ volume to below that of PSA (from 0.64 to 0.48). Linear regression analyses showed that, unlike PSA, both BPSA and free PSA displayed an age-independent relationship to TZ volume. The receiver operating characteristic curve (for TZ greater than 30 cm3) and binary logistic regression analyses showed that BPSA (area under the curve = 0.844) outperformed both free PSA (area under the curve = 0.799) and PSA (area under the curve = 0.749) in its ability to predict clinically significant TZ enlargement. CONCLUSIONS: In patients without prostate cancer, the serum concentration of BPSA displayed an age-independent, log-linear relationship to TZ volume and was a better predictor of prostatic enlargement than either PSA or free PSA. BPSA may also predict clinical parameters of BPH and is under evaluation as a marker of BPH progression and response to therapy.

Molecular diagnosis of prostate cancer.

The diagnosis, staging, and management of prostate cancer as we know it today is greatly dependent on our ability to measure serum prostate-specific antigen (PSA) concentration. Nevertheless, because serum PSA concentration, particularly when less than 10 ng/mL, reflects the presence of benign prostatic hyperplasia more often than cancer, there is a clear need for more specific prostate cancer markers. The most promising new markers for prostate cancer are the various molecular forms of free PSA. Mass spectrometry also is emerging as a potential tool in prostate cancer screening. Because it is unlikely that any one marker will have 100% sensitivity and specificity, as new serum markers are tested, nomograms that incorporate multiple independently predictive parameters for the detection of prostate cancer will become indispensable in our efforts to improve prostate cancer screening.

Predictors of prostate cancer after initial negative systematic 12 core biopsy.

PURPOSE: We determined the cancer detection rate at initial systematic 12 core (S12C) biopsy and identified features associated with cancer at repeat S12C biopsy after an initial negative S12C biopsy in patients with prostate specific antigen (PSA) parameters associated with a higher risk of prostate cancer. MATERIALS AND METHODS: Between February 1999 and June 2002, 841 patients underwent initial S12C biopsy. Of these patients 99 underwent repeat S12C biopsy after initial negative S12C because of a percent free-to-total PSA of 15.0 or less and/or a yearly PSA velocity of 0.75 ng/ml or greater. The association between parameters revealed by initial biopsy and cancer at repeat biopsy was assessed. RESULTS: Of the 99 patients 21 (21.2%) had cancer at repeat biopsy. Age (p = 0.01), PSA transitional zone density (p = 0.05), and high grade PIN at initial biopsy (p = 0.01) were associated with cancer at repeat biopsy. CONCLUSIONS: In this select group of patients with PSA parameters associated with a higher risk of prostate cancer the cancer detection rate after initially negative S12C biopsy was 21%. Patients with high grade PIN on initial biopsy, advanced age and higher PSA transition zone density are at increased risk for cancer at repeat biopsy. Larger prospective studies are required to confirm these results and construct a nomogram that determines the probability of finding prostate cancer at subsequent biopsy.

Improved detection of clinically significant, curable prostate cancer with systematic 12-core biopsy.

PURPOSE: While systematic 12-core (S12C) biopsy detects more cancers than sextant biopsy, to our knowledge the clinical significance of these additionally detected tumors has not been established. We studied pathological parameters of prostatectomy specimens from patients undergoing radical prostatectomy for prostate cancer detected with a S12C biopsy to determine the clinical significance of these cancers in comparison with sextant detected cancers. MATERIALS AND METHODS: A total of 179 consecutive patients undergoing radical prostatectomy for clinically localized prostate cancer detected by S12C biopsy were studied. The groups compared consisted of the sextant core subset of the S12C and the entire S12C set. Total tumor volume, Gleason score, organ confined status, surgical margin status, seminal vesicle invasion, lymph node involvement, and clinical significance of tumors detected by sextant and by S12C templates were compared. RESULTS: S12C biopsy detected a greater number of cancers scored as moderate (Gleason score 2 to 6) or high (Gleason score 7 or greater) grade, and cancers of all sizes regardless of organ confined status than the sextant cores alone (all p <0.05). S12C biopsy identified a greater number of biologically significant and insignificant tumors regardless of how they were defined. CONCLUSIONS: Compared with the sextant set S12C biopsy detects a significantly greater number of surgically curable, biologically significant tumors as well as those that might be considered clinically insignificant.

Six additional systematic lateral cores enhance sextant biopsy prediction of pathological features at radical prostatectomy.

PURPOSE: We evaluated the contribution of 6 additional systematically obtained, laterally directed biopsy cores to traditional sextant biopsy for the prediction of final pathological findings in the radical prostatectomy specimen. MATERIALS AND METHODS: We studied 178 consecutive patients with no history of prostate biopsy in whom prostate cancer was diagnosed during an initial systematic 12 core biopsy and who subsequently underwent radical prostatectomy. Of the systematic 12 cores we compared the subset of the 6 traditional sextant cores (S6C), the set of 6 laterally directed cores (L6C) and the complete 12 core set, which included the 6 traditional sextant and the 6 laterally directed cores. Biopsy Gleason score, number of positive cores, total cancer length and percent of tumor in the biopsy sets were examined for their ability to predict extracapsular extension, total tumor volume and pathological Gleason score. RESULTS: On univariable analyses the biopsy parameters of the complete 12 core set correlated more strongly with extracapsular extension and total tumor volume than the biopsy parameters of S6C or L6C. On multivariable analyses S6C and L6C were independent predictors of pathological features at prostatectomy. CONCLUSIONS: The addition of 6 systematically obtained, laterally directed cores to traditional sextant biopsy improved the ability to predict pathological features at prostatectomy by a statistically and prognostically significant margin. Preoperative nomograms that use data from a full complement of 12 systematic cores, specifying sextant and laterally directed biopsy cores, should demonstrate improved performance in predicting prostatectomy pathology.

Beyond prostate-specific antigen: new serologic biomarkers for improved diagnosis and management of prostate cancer.

The use of total prostate-specific antigen (tPSA) measurement has dramatically improved the ability to detect prostate cancer at earlier stages. However, as the number of men presenting with advanced disease (and high tPSA levels) has decreased, and given the fact that tPSA is highly reflective of benign prostatic hyperplasia, the need has emerged for novel biomarkers specifically associated with prostate cancer in order to improve predictive models. Several new biomarkers have shown promise, and studies continue to investigate the role of these markers in the detection, staging, and prognosis of prostate cancer. As new useful biomarkers continue to emerge, guidelines for their employment, as well as coordination of further research studies, are needed; a systematic, phased, nomogram-based model is a rational way to manage these efforts.

The addition of interleukin-6 soluble receptor and transforming growth factor beta1 improves a preoperative nomogram for predicting biochemical progression in patients with clinically localized prostate cancer.

PURPOSE: Several preoperative prostate cancer nomograms have been developed that predict risk of progression using pretreatment prostate-specific antigen (PSA) level, clinical stage, and biopsy Gleason grade. We describe the development and performance of a new nomogram. The nomogram adds new markers to the standard clinical predictors that reflect the biologic behavior of prostate cancer: pretreatment plasma levels of interleukin-6 soluble receptor (IL6SR) and transforming growth factor beta1 (TGF-beta1). PATIENTS AND METHODS: Between November 7, 1994 and December 22, 1997, 714 patients with stage cT1c to cT3a prostate cancer and no prior therapy were treated with radical prostatectomy at the Methodist Hospital, Houston TX. Plasma levels of IL6SR and TGF-beta1 were measured in banked preoperative plasma. With these data, a nomogram was developed to predict the probability of PSA progression within 5 years of surgery. The nomogram was validated with bootstrapping to assess its discrimination and calibration performance. RESULTS: In the multivariable Cox model, PSA (P =.004), IL6SR (P <.001), TGF-beta1 (P <.001), primary Gleason grade (P <.002), and secondary Gleason grade (P =.029) were associated with PSA progression, whereas clinical stage (P =.696) was not. The nomogram seemed to be well calibrated and had a bootstrap-corrected area under the receiver operating characteristic curve (ie, concordance index) of 0.83. For comparison, a nomogram that omitted IL6SR and TGF-beta1 achieved a concordance index of only 0.75. CONCLUSION: We found that pretreatment plasma levels of IL6SR and TGF-beta1 improved the ability to predict biochemical progression by a prognostically substantial margin. A nomogram including the pretreatment levels of these molecular markers, along with standard clinical markers, has been developed and internally validated.

Biochemical staging of prostate cancer.

PSA continues to be one of the most effective and widely used cancer screening tools available. Its popularity in prostate cancer screening, however, has eroded its usefulness in the staging of this disease. As more men are screened every year on a routine basis with DRE and PSA, the average PSA at diagnosis has drifted down to well below 10 ng/mL in many centers, including ours. This trend is likely to accelerate, as a PSA cut off for prompting biopsy of the prostate of 2.5 ng/mL gains more widespread acceptance. The recent realization that, at these levels, serum PSA is more reflective of the presence of BPH than of the extent of cancer and, therefore, does not provide additional staging information, has renewed the search for new biochemical markers that are capable of predicting prostate cancer stage and prognosis. Because of the heterogeneity of this disease, it is unlikely that a single biochemical marker that is capable of accurately staging all prostate cancer patients will be found. For this reason, nomograms that are capable of integrating various parameters to predict stage and prognosis will remain indispensable. As new biochemical markers that provide independent predictive information about stage or prognosis are identified, they can be incorporated into currently available nomograms. Of the biochemical markers discussed in this article, IL-6sR and TGF-beta1 are the most promising. By incorporating them into a preoperative nomogram designed to predict PSA recurrence, we found that they improved the ability to predict biochemical recurrence by a statistically and clinically significant margin. The ability to stage prostate cancer and predict response to therapy has improved dramatically over the last 3 decades. Nevertheless, there is still a need for new biochemical markers that will improve the ability to predict an individual patient's stage and response to therapy. Incorporating these new markers into nomograms will enhance the ability to provide optimal care for each prostate cancer patient.

Early management of prostate cancer: how to respond to an elevated PSA?

Support for prostate cancer screening efforts is provided by observational studies reporting decreases in prostate cancer-specific mortality in areas where screening is performed with digital rectal exam (DRE) and measurement of serum prostate-specific antigen (PSA) levels. The combination of PSA and DRE is an excellent cancer-screening tool with sensitivity and positive predictive value superior to that of mammography and breast exam. Use of percent free PSA further improves the specificity of PSA testing, particularly in the range of 4-10 ng/ml, at which most false positive PSA tests occur. Men older than 50 with a >10-year life expectancy should be considered for prostate cancer screening. Those with an abnormal DRE or a PSA above 4 ng/ml should be referred to a urologist for further discussion of the risks and benefits of a prostate biopsy. Furthermore, those with a significant change in either DRE or PSA results, or those at higher risk for prostate cancer with a PSA level above 2.5 ng/ml, should also be referred for evaluation.

Sural Nerve Interposition Grafting during Radical Prostatectomy.

In 1997, autologous sural nerve grafting to reconstruct bilaterally resected cavernosal nerves was successfully performed in patients undergoing radical retropubic prostatectomy. After 12 months, one third of these patients had erections sufficient for intercourse. Since that time, patients who have had neurovascular bundle resection and sural nerve grafting have continued to show promising results. For example, within one large cohort of men who had unilateral, nerve-sparing radical prostatectomy, significantly more men who had sural nerve grafting regained potency, and did so in less time, than men who did not have grafting. More importantly, however, with better predictions of the presence of extracapsular disease, nerve-sparing surgery can be performed more selectively, reserving wide resection and sural nerve grafting for patients likely to have extracapsular extension. A multicenter, randomized clinical trial is needed to substantiate the positive outcomes observed with sural nerve grafting.