Impact of the US Preventive Services Task Force Grade D Recommendation: Assessment of Evaluations for Elevated Prostate-specific Antigen and Prostate Biopsies in a Large Urology Group Practice Following Statement Revision

On October 7, 2011, the United States Preventive Services Task Force (USPSTF) released their evidence statement and grade D recommendation against prostate-specific antigen (PSA)-based prostate cancer screening. Using a time series design, we assessed the effect of this recommendation upon evaluations for elevated PSA levels and prostate biopsies in our large urology group practice. We found that, despite a 24.1% increase in total visits, the 32 urologists in our practice completed 16.4% fewer evaluations for elevated PSA levels (317 fewer evaluations per month; P = .017) and 21.4% fewer prostate biopsies (42 fewer biopsies per month; P = .001) in the 2 years following the USPSTF grade D recommendation.Key words: Prostate-specific antigen, Prostate cancer screening, Prostate biopsies, United StatesProstate cancer is the most common noncutaneous malignancy in American men. In the United States in 2015, approximately 220,800 men will be diagnosed with prostate cancer and 27,540 men will die from the disease.¹In 1986, the US Food and Drug Administration approved prostatespecific antigen (PSA) testing for monitoring disease progression in men previously diagnosed with prostate cancer.² In 1991, Catalona and colleagues³ published their findings that, when coupled with digital rectal examination and ultrasound, serum PSA measurement improved the detection of prostate cancer.Aiming to clarify the effect of PSA-based prostate cancer screening upon prostate cancer mortality, two large randomized trials of screening matured in 2009: the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)⁴ and the European Randomized Study of Screening for Prostate Cancer (ERSPC).⁵ After 7 to 10 years of follow-up, the PLCO trial found no difference in prostate cancer mortality between men randomized to annual PSA testing and digital rectal examination versus usual care.⁴ In the ERSPC trial, PSA-based screening reduced the rate of death from prostate cancer by 20% at 9 years median follow-up. This reduction in prostate cancer mortality was associated with a high risk of over-diagnosis: 1410 men needed screening and 48 additional cases of prostate cancer required treatment to prevent 1 death from prostate cancer.⁵Using these two large studies as their evidence foundation for the benefits of early detection and treatment of prostate cancer, the United States Preventive Services Task Force (USPSTF) determined that the harms of PSA-based prostate cancer screening outweighed the benefits. On October 7, 2011, the USPSTF published their evidence statement and draft recommendation against PSA-based prostate cancer screening. ⁶ Extensive media coverage and national discussion ensued, with many publically disagreeing with the Task Force’s draft recommendation. ^7–9 In May 2012, the USPSTF finalized their grade D recommendation: PSA-based prostate cancer screening should be discouraged.¹⁰Although multiple screening guidelines exist that differ from those of the USPSTF,^11–13 primary care physicians are historically most influenced by the USPSTF recommendations. ¹⁴ In a study of primary care providers from Johns Hopkins Community Physicians, a university-affiliated practice including 26 outpatient sites in 11 Maryland counties, following release of the USPSTF draft recommendation against PSA-based prostate cancer screening, fewer than 50% agreed with the new recommendation, suggesting the change may encounter significant barriers to adoption.¹⁵ Consistent with this observation, various effects of the USPSTF recommendation upon the number of PSA tests performed, ^16–19 evaluations for elevated PSA levels,²⁰ and prostate biopsies completed^20–22 have been reported in the literature since 2012. Based on our clinical observations, we hypothesized that the number of evaluations for elevated PSA levels and number of prostate biopsies performed in our community-based, large urology group practice would decrease significantly following the publication of the USPSTF draft recommendation against prostate cancer screening.     

Focal Laser Ablation for Localized Prostate Cancer: Principles,Clinical Trials,and Our Initial Experience

Focal therapy of prostate cancer is an evolving treatment strategy that destroys a predefined region of the prostate gland that harbors clinically significant disease. Although long-term oncologic control has yet to be demonstrated, focal therapy is associated with a marked decrease in treatment-related morbidity. Focal laser ablation is an emerging modality that has several advantages, most notably real-time magnetic resonance imaging (MRI) compatibility. This review presents the principles of laser ablation, the role of multiparametric MRI for delineating the site of significant prostate cancer, a summary of published clinical studies, and our initial experience with 23 patients, criteria for selecting candidates for focal prostate ablation, and speculation regarding future directions.Key words: Laser ablation, Prostate cancer, Focal therapy, Targeted therapyProstate cancer is the most common solid organ malignancy and the second most common cause of cancer death among men living in the Western world.¹ Widespread prostate-specific antigen (PSA) testing and decreased thresholds for prostate biopsy have led to both a reduction in the proportion of men diagnosed with advanced disease and disease-specific mortality. The consequence of widespread PSA screening has been a dramatic increase in both the detection of low-risk disease and the proportion of men diagnosed with prostate cancer undergoing radical prostatectomy (RP) or radiation therapy (RT).² In many cases, the complications associated with treating low-risk disease by RP or RT outweigh the benefits.^3,4 Although active surveillance (AS) is an appealing alternative for managing low-risk disease, it potentially decreases long-term survival rates.⁵ Due to the unreliability of disease risk stratification at the time of diagnosis, 14% to 41% of men assigned to AS will cross over to RP or RT due to upgrading or upstaging.⁶There is increasing evidence that multiparametric magnetic resonance imaging (mpMRI) localizes the site(s) of clinically significant prostate cancer prior to prostate biopsy.⁷ These suspicious MRI focal abnormalities can be biopsied directly in the MRI unit or under transrectal ultrasound (TRUS) guidance using software that co-registers and fuses the MRI and ultrasound (US) images.⁸ In many cases, MRI image-guided biopsy identifies a single clinically significant cancer. Although prostate cancer is typically a multifocal disease, the index, or dominant, lesion is typically predictive of extraprostatic extension and disease progression.^9–11 The majority of the secondary tumor sites are composed of small Gleason 6 disease, which represent no immediate threat.¹² It is theoretically possible to focally ablate only the index lesion, thereby achieving oncologic control while minimizing treatment-related morbidity by minimizing collateral damage to adjacent structures.Focal ablation of prostate cancer is an evolving treatment strategy that destroys a predefined region (or target) of the prostate that harbors the clinically significant cancer. A number of energy sources have been investigated for focal ablation of the prostate, including cryotherapy,¹³ high-intensity focused ultrasound (HIFU),¹⁴ photodynamic therapy,¹⁵ and laser ablation.¹⁶ Although long-term oncologic control has yet to be demonstrated, all of these targeted ablative options are associated with marked decrease in treatment-related complications. One of the advantages of laser technology is that the ablation can be performed with real-time MRI imaging. Because the target lesion are almost always defined by the MRI, laser ablation is currently the most accurate way to deliver ablative energy to the intended target. Other advantages of laser ablation include its homogeneous tissue necrosis, relatively low cost, and wide availability.¹⁷ MRI-guided focal ablation allows treatment monitoring using MR thermometry and real-time visualization of the targeted treatment zone.^18,19This review presents the principles of laser ablation, the role of mpMRI for delineating the site of significant prostate cancer, a summary of published clinical studies and the New York University Langone Medical Center (NYULMC)/Sperling Prostate Cancer Center experience on focal laser ablation of prostate cancer, criteria for selecting candidates for focal prostate ablation, and speculation regarding future directions of focal laser ablation for the treatment of localized prostate cancer.     

Activation of an Olfactory Receptor Inhibits Proliferation of Prostate Cancer Cells

Olfactory receptors (ORs) are expressed not only in the sensory neurons of the olfactory epithelium, where they detect volatile substances, but also in various other tissues where their potential functions are largely unknown. Here, we report the physiological characterization of human OR51E2, also named prostate-specific G-protein-coupled receptor (PSGR) due to its reported up-regulation in prostate cancer. We identified androstenone derivatives as ligands for the recombinant receptor. PSGR can also be activated with the odorant β-ionone. Activation of the endogenous receptor in prostate cancer cells by the identified ligands evoked an intracellular Ca²⁺ increase. Exposure to β-ionone resulted in the activation of members of the MAPK family and inhibition of cell proliferation. Our data give support to the hypothesis that because PSGR signaling could reduce growth of prostate cancer cells, specific receptor ligands might therefore be potential candidates for prostate cancer treatment.Excessive signaling by G-protein-coupled receptors (GPCRs)³ such as endothelin A receptor (1), bradykinin 1 receptor (2), follicle-stimulating hormone receptor (3), and thrombin receptor (4, 5) is known to occur in prostate cancers due to strong overexpression of the respective receptors. Activation of some of these GPCRs results in androgen-independent androgen receptor activation, thus promoting the transition of prostate cancer cells from an androgen-dependent to an androgen-independent state (6, 7).The prostate-specific G-protein-coupled receptor (PSGR) is a class A GPCR that was initially identified as a prostate-specific tumor biomarker (8–10). It is specifically expressed in prostate epithelial cells, and its expression increases significantly in human prostate intraepithelial neoplasia and prostate tumors, suggesting that PSGR may play an important role in early prostate cancer development and progression (9, 11). Although expression of the human PSGR was found to be prostate-specific (10, 12), mRNA can also be detected in the olfactory zone and the medulla oblongata of the human brain (12). Human PSGR shares 93% amino acid homology to the respective mouse and rat homologues, which are also expressed in the brain (12). Interestingly, PSGR has numerous sequence motifs in common with the large superfamily of olfactory receptors (ORs), which build the largest class of human GPCRs and allow the recognition of a wide range of structurally diverse molecules in the nasal epithelium (13–15). Recently, also the steroid hormones androstenone and androstadienone were identified as OR ligands (16). In addition to their role in the sensory neurons of the nose, ORs have been found in different tissues throughout the body (17, 18). Their function(s) in these extranasal locations are questionable except for in a few cases where functional studies have been performed in spermatozoa (19, 20) and in enterochromaffin cells of the gastrointestinal tract (21).Here, we report the identification of steroid ligands of heterologously expressed PSGR and investigate the functional relevance of PSGR expression in prostate tissue. Steroid hormones elicited rapid Ca²⁺ responses in the LNCaP prostate cancer cell line and in primary human prostate epithelial cells. Moreover, activated PSGR causes phosphorylation of p38 and stress-activated protein kinase/c-Jun NH₂-terminal kinase (SAPK/JNK) mitogen-activated protein kinases (MAPKs), resulting in reduced proliferation rates in prostate cancer cells.     

Finding the Wolf in Sheep’s Clothing: The 4Kscore Is a Novel Blood Test That Can Accurately Identify the Risk of Aggressive Prostate Cancer

Better biomarkers that can discriminate between aggressive and indolent phenotypes of prostate cancer are urgently needed. In the first 20 years of the prostate-specific antigen (PSA) era, screening for prostate cancer has successfully reduced prostate cancer mortality, but has led to significant problems with overdiagnosis and overtreatment. As a result, many men are subjected to unnecessary prostate biopsies and overtreatment of indolent cancer in order to save one man from dying of prostate cancer. A novel blood test known as the 4Kscore® Test (OPKO Lab, Nashville, TN) incorporates a panel of four kallikrein protein biomarkers (total PSA, free PSA, intact PSA, and human kallikrein-related peptidase 2) and other clinical information in an algorithm that provides a percent risk for a high-grade (Gleason score ≥ 7) cancer on biopsy. In 10 peer-reviewed publications, the four kallikrein biomarkers and algorithm of the 4Kscore Test have been shown to improve the prediction not only of biopsy histopathology, but also surgical pathology and occurrence of aggressive, metastatic disease. Recently, a blinded prospective trial of the 4Kscore Test was conducted across the United States among 1012 men. The 4Kscore Test replicated previous European results showing accuracy in predicting biopsy outcome of Gleason score ≥ 7. In a recent case-control study nested within a population-based cohort from Västerbotten, Sweden, the four kallikrein biomarkers of the 4Kscore Test also predicted the risk for aggressive prostate cancer that metastasized within 20 years after the test was administered. These results indicate that men with an abnormal PSA or digital rectal examination result, and for whom an initial or repeat prostate biopsy is being considered, would benefit from a reflex 4Kscore Test to add important information to the clinical decision-making process. A high-risk 4Kscore Test result may be used to select men with a high probability of aggressive prostate cancer who would benefit from a biopsy of the prostate to prevent an adverse and potentially lethal outcome from prostate cancer. Men with a low 4Kscore Test result may safely defer biopsy.Key words: Prostate cancer, Biomarker, High-grade prostate cancer, ScreeningProstate cancer is the most common cancer in men in the United States, accounting for an estimated 27% of all newly diagnosed cancers in 2014.¹ Since the advent of screening for prostate cancer with serum prostate-specific antigen (PSA), we have seen a significant decline in prostate cancer mortality.¹ Randomized clinical trials have reported a 20% to 40% reduction in death from prostate cancer in men undergoing routine screening compared with those who are not screened.^2,3 However, these trials, and a trial showing little difference between opportunistic and systematic screening,⁴ have raised the concern for overdiagnosis and overtreatment of indolent prostate cancer. The fundamental concern is that an overwhelming number of men are subjected to interventions such as prostate biopsy in order to prevent one man’s death from prostate cancer.^2,3Prostate biopsy is an invasive procedure with significant complications, such as bleeding, urinary retention, and life-threatening infection. A recent population-based study from Ontario, Canada, revealed a fourfold increase to 4.1% for the rate of hospital admissions after prostate biopsy from 1996 to 2005, with 72% of admissions being due to infection.⁵ These risks, combined with the enormous anxiety involved in undergoing the procedure, present a significant burden to any man considering prostate cancer screening.Today, most men diagnosed with prostate cancer have a tumor that is unlikely to pose a threat to their life expectancies. A recent systematic analysis suggested that up to 60% of prostate cancers diagnosed in contemporary studies can be safely observed without a need for immediate intervention.⁶ However, in the United States, because of the concern for possible undergrading of prostate cancer due to biopsy sampling error, 90% of men diagnosed with prostate cancer undergo treatment and approximately 66% will be confirmed to have indolent Gleason score 6 prostate cancer,⁷ suggesting a significant problem with overtreatment. Although treatment for localized prostate cancer provides excellent cancer control,^8,9 it comes at a significant detriment to health-related quality of life (HRQoL). Previous studies have reported significant changes in HRQoL after primary treatment for prostate cancer, primarily in the domains of sexual and urinary function and bother.^10–12 Given the physical and psychological burden of these secondary adverse events, many government agencies and patients are beginning to question the risks and benefits of prostate cancer screening and treatment.¹³The United States Preventive Services Task Force recently advised against routine screening for prostate cancer, claiming that the risks of screening outweigh the benefits.¹³ However, 20% to 30% of men who are diagnosed with prostate cancer are found to have high-grade disease at presentation¹⁴; without screening, these men would lose their opportunity for cure. It is clear that new biomarkers or tests that promote the detection of both indolent and aggressive prostate cancer are unlikely to be helpful. We need tests that focus on the detection of aggressive tumors, not the indolent ones that are better left alone. Aggressive prostate cancer, for purposes of this review, is defined as cancer with a Gleason score ≥ 7 and tumors that are most likely to progress to metastatic disease and death. Targeted detection of aggressive prostate cancer would allow urologists to diagnose and treat those men most likely to benefit from aggressive intervention to avoid premature death. Conversely, those men harboring non-life-threatening disease would be able to avoid unnecessary interventions. The 4Kscore® Test (OPKO Lab, Nashville, TN) is a new blood test that accurately identifies the risk of aggressive prostate cancer. The 4Kscore Test plays an important clinical role as a reflex test prior to proceeding with initial prostate biopsy in men with an elevated PSA level or abnormal digital rectal examination (DRE) results, or after a prior negative biopsy and persistently abnormal PSA levels.     

Identification of Differentially Expressed Proteins in Direct Expressed Prostatic Secretions of Men with Organ-confined Versus Extracapsular Prostate Cancer

Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.Prostate cancer is the most common malignancy to affect men in the Western world, but only 15–20% of these men will present with aggressive, lethal disease (1, 2) whereas the majority of patients will die of other causes. Although the implementation of large-scale screening for prostate cancer using serum prostate-specific antigen (PSA) has dramatically improved early detection of disease, unnecessary biopsies and patient overtreatment are becoming increasingly evident (2, 3). Consequently, there has been a shift in emphasis away from detection of prostate cancer and toward identification of lethal disease. Currently, Gleason grading is considered to be one of the best outcome predictors; however, patients with Gleason 7 tumors are in the clinical “gray zone,” whereby the predictive ability of Gleason grading is mixed (4, 5). A recent study constructed a 157-gene signature based on the comparison of Gleason score ≤6 and ≥8 patients, and could show that their panel could predict lethality in the cohort of Gleason 7 patients (5). Nonetheless, the development and large-scale implementation of prognostic markers of prostate cancer has been hampered by numerous factors owing, in part, to the heterogeneous and multifocal nature of the disease (6). Although the widely used Gleason grading system attempts to control for heterogeneity of the glands and multifocality of cancerous lesions by summing the 2–3 most commonly observed histological patterns via inspection of multiple (typically 8–12) core biopsies, cancerous foci are still often missed (2, 6) providing only partial information that can lead to imprecise diagnoses and prognoses. Pathologic staging remains the gold standard for disease staging and risk assessment (7, 8); however, this process lacks timeliness in discriminating organ-confined from extracapsular disease. Indeed, one-third of individuals with nonorgan-confined disease are identified only after surgery (9). Furthermore, ∼35% of men treated with radical prostatectomy with curative intent subsequently develop biochemical recurrence (10–13) and the mean time from surgery to recurrence is 3.5 years (4). Significant risk factors for time to prostate-specific mortality following biochemical recurrence after radical prostatectomy are PSA doubling time, pathological Gleason score, and time from surgery to biochemical recurrence (4). Estimates place the percent of lethal cases at 20–25% of all patients that show biochemical recurrence, suggesting that nearly 75–80% of patients in this group may be overtreated (14).There is an emerging trend toward recruitment of men with perceived low-risk disease to an “active surveillance” monitoring approach. This is based on the supposition that most prostate cancers are slow growing, and that the more aggressive forms can be identified during a period of observation with little increased risk of death. Although a consensus may not exist for defining the disease stage where active surveillance is warranted, there is considerable agreement that men who have a PSA level less than 10 ng/ml, impalpable disease (clinical stage T1c) and only 1 biopsy core out of 12 or more that show Gleason 6 cancer are most likely to harbor indolent disease (15). Even so, these candidates for active surveillance will still contain individuals who will have disease progression and die from their cancer. Thus, despite efforts to recruit individuals to active surveillance protocols, overtreatment of prostate cancer is fueled by the lack of reliable means to accurately discriminate between men with clinically indolent prostate cancer from those with more aggressive disease (16, 17). This inability to accurately predict prostate cancer aggressiveness based solely on standard clinicopathologic features clearly underscores the need to explore the ability of additional biomarkers to enhance outcome prediction for men with prostate cancer. Furthermore, it is important to acknowledge that a single biomarker alone is unlikely to have sufficient prognostic power; rather, the integration of a panel of biomarkers hold the promise for improved prostate cancer detection and prognosis (2).Fluids that are proximal to the prostate are attractive sources of potential prostate cancer biomarkers (2, 18), as they house secreted proteins and sloughed cells that provide a presumably more comprehensive assessment of the organ and extent of disease. Further, fluids such as urine are clinically favorable for their ease of collection, the volume and frequency at which they can be obtained, and their adaptability to routine clinical assays. Prostate-proximal fluids include seminal fluid, semen, and expressed prostatic secretions (EPS)¹. Here, we focus on the analysis of EPS as our biological specimen, using direct-EPS samples for the discovery of candidate prognostic biomarkers and both direct-EPS and pooled EPS-urines derived from independent sets of patients for candidate biomarker verification. Direct-EPS is a prostatic fluid that is collected from patients undergoing prostatectomy by massaging the organ and expelling 0.5–1 ml of the fluid just prior to surgical removal. It was chosen as our discovery fluid as it is expected to house prostate-secreted proteins at a higher concentration and purity, and we have developed a workflow for the in-depth proteomic analysis of this fluid (19). Following discovery proteomics in 16 clinically stratified direct-EPS samples, verification studies were performed using independent sample sets of direct-EPS. Next, we focused our attention on the verification and quantitative analysis of candidate proteins in pooled EPS-urines. Before EPS-urine collection, men undergo digital rectal examination (DRE), often as part of a routine procedure, which causes direct-EPS to be expelled from the prostate and subsequently voided in urine. Because EPS-urine can be collected with substantial ease and in greater volumes and frequencies than direct-EPS, much attention has been paid to this fluid as a valuable resource of prostate cancer biomarkers amenable to routine clinical analysis. Following the recent FDA approval of the EPS-urine assay for prostate cancer gene 3 (PCA3), standardized clinical collection protocols will be widely implemented and easier access to this fluid is expected. Moreover, we have recently identified a number of prostate-enriched proteins in EPS-urine by comparing its proteome to a urine background (20).The present study used multidimensional protein identification technology (MudPIT) coupled with bioinformatics to first catalog and comparatively analyze the direct-EPS proteomes from a small cohort of patients with extracapsular versus organ-confined prostate cancers. A semiquantitative algorithm based on spectral counts (QSpec) (21) and an integrative data mining strategy led to the selection of a number of putative biomarkers that were verified by Western blotting in direct-EPS. Lastly, to demonstrate accurate quantitative measurements of verified candidates in EPS-urine, a pilot study utilizing SRM-MS was undertaken as a proof-of-concept.     

Emerging Therapeutic for the Treatment of Skeletal-related Events Associated With Metastatic Castrate-resistant Prostate Cancer

Prostate cancer is the most prevalent cancer in US and European men and the second leading cause of cancer death in those populations. It is somewhat unique in that nearly all patients who succumb to the disease will ultimately develop bone metastasis. Morbidity from bone metastasis-referred to as skeletal-related events, which include fractures, cord compression, radiation to bone, and surgery to bone—leads to significant costs and impaired quality of life. This article reviews three agents and the roles they play in the ever-changing armamentarium of treatments for metastatic castrate-resistant prostate cancer (mCRPC). The potential benefits of these agents are discussed, as well as the continuing use of these agents and their earlier introduction in the patient with progressive mCRPC with bone metastasis.Key words: Metastatic castrate-resistant prostate cancer, Skeletal-related events, Bone metastasis, Zoledronic acid, Denosumab, Radium Ra 223 dichlorideProstate cancer is the most prevalent cancer in US and European men and the second leading cause of cancer death in those populations. It is somewhat unique in that nearly all patients who have the disease will ultimately develop bone metastasis.¹ Morbidity from bone metastasis—referred to as skeletal—related events (SREs), which include fractures, cord compression, radiation to bone, and surgery to bone-leads to significant costs and impaired quality of life. An estimated 241,740 men are diagnosed with prostate cancer each year in the United States¹; between 9.5% and 17.8% of these patients have M0 + M1 castrate-resistant prostate cancer (CRPC).^2,3Skeletal tumor burden and fracture are both independent predictors of death in men with metastatic CRPC (mCRPC).^2,3 In addition, pain is an independent prognosticator for death⁴; thus, agents that reduce pain may improve quality as well as quantity of life. In the past decade, three new agents have been approved in the United States for the treatment and/or prevention of SREs in men with mCRPC. However, urologists continue to under-treat this condition.⁵ A recent clinical trial that screened a large population of men thought to have CRPC without metastasis found nearly one third of patients to have metastatic prostate cancer.⁶ And a recent large clinical trial in men with mCRPC, most of whom had bone metastases, showed fewer than 50% of patients were receiving a bisphosphonate.⁷This article reviews these three agents and the new roles they play in the ever-changing armamentarium of treatments for mCRPC. The potential benefits of these agents are discussed, as well as the continuing use of these agents and their earlier introduction in the patient with progressive mCRPC with bone metastasis.     

Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.Colorectal cancer (CRC)¹ is the second most prevalent cancer in the western world. The development of this disease takes decades and involves multiple genetic events. CRC remains a major cause of mortality in developed countries because most of the patients are diagnosed at advanced stages because of the reluctance to use highly invasive diagnostic tools like colonoscopy. Actually only a few proteins have been described as biomarkers in CRC (carcinoembryonic antigen (CEA), CA19.9, and CA125 (1–3)), although none of them is recommended for clinical screening (4). Proteomics analysis is actively used for the identification of new biomarkers. In previous studies, the use of two-dimensional DIGE and antibody microarrays allowed the identification of differentially expressed proteins in CRC tissue, including isoforms and post-translational modifications responsible for modifications in signaling pathways (5–8). Both approaches resulted in the identification of a collection of potential tumoral tissue biomarkers that is currently being investigated.However, the implementation of simpler, non-invasive methods for the early detection of CRC should be based on the identification of proteins or antibodies in serum or plasma (9–13). There is ample evidence of the existence of an immune response to cancer in humans as demonstrated by the presence of autoantibodies in cancer sera. Self-proteins (autoantigens) altered before or during tumor formation can elicit an immune response (13–19). These tumor-specific autoantibodies can be detected at early cancer stages and prior to cancer diagnosis revealing a great potential as biomarkers (14, 15, 20). Tumor proteins can be affected by specific point mutations, misfolding, overexpression, aberrant glycosylation, truncation, or aberrant degradation (e.g. p53, HER2, NY-ESO1, or MUC1 (16, 21–25)). In fact, a number of tumor-associated autoantigens (TAAs) were identified previously in different studies involving autoantibody screening in CRC (26–28).Several approaches have been used to identify TAAs in cancer, including natural protein arrays prepared with fractions obtained from two-dimensional LC separations of tumoral samples (29, 30) or protein extracts from cancer cells or tissue (9, 31) probed by Western blot with patient sera, cancer tissue peptide libraries expressed as cDNA expression libraries for serological screening (serological analysis of recombinant cDNA expression libraries (SEREX)) (22, 32), or peptides expressed on the surface of phages in combination with microarrays (17, 18, 33, 34). However, these approaches suffer from several drawbacks. In some cases TAAs have to be isolated and identified from the reactive protein lysate by LC-MS techniques, or in the phage display approach, the reactive TAA could be a mimotope without a corresponding linear amino acid sequence. Moreover, cDNA libraries might not be representative of the protein expression levels in tumors as there is a poor correspondence between mRNA and protein levels.Protein arrays provide a novel platform for the identification of both autoantibodies and their respective TAAs for diagnostic purposes in cancer serum patients. They present some advantages. Proteins printed on the microarray are known “a priori,” avoiding the need for later identifications and the discovery of mimotopes. There is no bias in protein selection as the proteins are printed at a similar concentration. This should result in a higher sensitivity for biomarker identification (13, 35, 36).In this study, we used commercially available high density protein microarrays for the identification of autoantibody signatures and tumor-associated antigens in colorectal cancer. We screened 20 CRC patient and control sera with protein microarrays containing 8000 human proteins to identify the CRC-associated autoantibody repertoire and the corresponding TAAs. Autoantibody profiles that discriminated the different types of CRC metastasis were identified. Moreover a set of TAAs showing increased or decreased expression in cancer cell lines and paired tumoral tissues was found. Finally an ELISA was set up to test the ability of the most immunoreactive proteins to detect colorectal adenocarcinoma. On the basis of the antibody response, combinations of three antigens, PIM1, MAPKAPK3, and ACVR2B, showed a great potential for diagnosis.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery

The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis.Despite many advances in the treatment of cancer, early detection and tumor removal remains the best prospect for overcoming disease. Ovarian cancer is an excellent example of the potential prognostic value of early detection because diagnosis at a localized stage has a 5-year survival rate of 93%. However, only 19% of cases are diagnosed at this stage, and by the time the disease has evolved to an advanced stage, the 5-year survival rate drops to 31% (1).Much effort has been expended to find early detection markers of ovarian cancer, and some success has been achieved. Most notable is CA125, the only approved marker for the detection of recurrence of ovarian cancer (2). Other leading targets are mesothelin and HE4, which have been examined by several groups for their efficacy as early detection markers (3–8). Nevertheless, several conditions necessitate the discovery of more specific and sensitive ovarian cancer markers: the heterogeneity of this disease, the ambiguity of its symptoms, its low incidence in the general population, and the low sensitivity and specificity of currently available markers.One of the difficulties in finding markers in blood is the complexity of the plasma/serum proteome, estimated in the tens to hundreds of thousands of proteins, as well as its large range in constituent protein concentrations, which can span 12 orders of magnitude (9). However, along with its easy accessibility, the fact that blood is in contact with virtually every tissue and contains tissue- and tumor-derived proteins makes it a preferred source for disease biomarker discovery.Our previous results (10, 11) and those of others (12–14) using high density, full-length IgG antibody microarrays to validate and discover cancer serum biomarkers demonstrated that this platform is valuable for simultaneously comparing the levels of hundreds of proteins on dozens of serum samples from cancer patients and healthy controls. We confirmed overexpression of CA125, mesothelin, and HE4 in ovarian cancer samples using this high density microarray platform, validating our array methodology for measurement of cancer serum biomarkers and yielding new putative biomarkers for this disease (10, 11).Previously reported approaches are typically limited to a few hundred antibodies. The methodology reported here allows us to exploit the specific advantages of antibodies as high affinity capture reagents to detect differential expression of thousands of tumor biomarkers using a diverse (2 × 10⁸ binding agents) single-chain variable fragment antibody (scFv)¹ library for detection of ovarian cancer markers in serum, tumor cyst fluid, and ascites fluid. Our results build on previous reports of phage display library microarrays to discover autoantibody (15–18) and other protein (12, 19, 20) cancer biomarkers. Our scFv are high affinity capture reagents consisting of the variable regions of human antibody heavy and light chains joined by a flexible linker peptide. These recombinant antibodies are able to recognize a wide variety of antigens, including many previously thought difficult, such as self-antigens and proteins that are not normally immunogenic in animals (21–24). Using a highly diverse recombinant antibody library, one has the ability to overcome the complexity of the serum proteome. It has been calculated that for an immune repertoire to be complete (at least one antibody in the repertoire has reasonable affinity for every epitope possible in nature) it requires a diversity of at least 10⁶ antibodies (25). The reported diversity of our scFv library exceeds this value by 100-fold (21).To enrich for antibodies that differentiate disease status, we performed a selection or panning of the naïve library for proteins that are differentially expressed in cyst fluid, ascites fluid, or serum of cancer patients with respect to healthy serum. We printed this sublibrary on activated hydrogel slides that were queried with three different sets of labeled case and control sera to further select those that discriminate cancer status in a statistically significant manner. Next, we identified some of the targets that bind to the individual scFv using high density nucleic acid programmable protein arrays (NAPPAs) expressing a total of over 7000 proteins. Finally, we validated the effectiveness of the selection process by confirming overexpression of these targets in cancer serum, cyst fluid, and ascites fluid as well as in tumor sections.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]