The Rationale for Optimal Combination Therapy With Sipuleucel-T for Patients With Castration-resistant Prostate Cancer

Immunotherapy encourages the recipient’s own immune response to destroy cancer cells, and current evidence suggests that immunotherapies may be most beneficial in early metastatic castration-resistant prostate cancer (mCRPC). Sipuleucel-T is the first therapeutic cancer vaccine to be approved by both the US Food and Drug Administration and European Medicines Agency for the treatment of asymptomatic or minimally symptomatic mCRPC. Combining immunotherapy with other treatments may have potent anticancer effects; cytoreductive therapies can release tumor antigens and promote a proinflammatory environment that could augment immunotherapies. However, some cytoreductive agents or coadministered drugs may be immunosuppressive. Understanding these interactions between different mCRPC treatment modalities may offer further potential to improve patient outcomes.Key words: Combination therapy, Prostate cancer, Sipuleucel-TImmunotherapy has emerged as a powerful tool against prostate cancer, in addition to surgery, radiotherapy, hormone therapy, and chemotherapy. For 30 years, investigators tried to rebalance the compromised immune system in patients with urologic cancers using a number of different agents.^1,2 In April 2010, the autologous cellular immunotherapy sipuleucel-T became the first therapeutic cancer vaccine to be approved by the US Food and Drug Administration (FDA).³ This therapy targets the prostatic acid phosphatase (PAP) and has been indicated for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC), based on results from three randomized, controlled, phase 3 studies.^3–6 Recently, sipuleucel-T was also approved by the European Medicines Agency (EMA) for the treatment of asymptomatic or minimally symptomatic mCRPC in men in whom chemotherapy is not yet clinically indicated.⁷Although this immunotherapy has been shown to extend overall survival (OS),⁵ sequencing or combining immunotherapy with other treatments for mCRPC has the potential to further improve outcomes.^8,9 However, before immunotherapy-based combination regimens can be integrated into clinical practice, it is critical to have a better understanding of the interactions between these different modalities.     

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.     

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.     

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.     

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.     

Fertility Preservation Options for Men and Women With Cancer

Approximately 0.2% of Americans aged 20 to 39 years are childhood cancer survivors. Advances in cancer detection and therapy have greatly improved survival rates for young cancer patients; however, treatment of childhood cancers can adversely impact reproductive function. Many cancer patients report a strong desire to be informed of existing options for fertility preservation and future reproduction prior to initiation of gonadotoxic cancer therapies, including surgery, chemotherapy, and radiotherapy. This article discusses, in detail, the effects of cancer treatment on fertility in men and women, and outlines both current and experimental methods of fertility preservation among cancer patients.Key words: Fertility preservation, Childhood cancer, Sperm cryopreservation, Testicular tissue cryopreservation, Spermatogonial stem cell cryopreservation, Embryo cryopreservation, Oocyte cryopreservation, Ovarian tissue cryopreservationIn 2014, an estimated 15,780 new cancer cases were diagnosed among children and adolescents younger than age 20 years, resulting in 1960 deaths. In addition, 1 in 285 children will be diagnosed with cancer before age 20, and approximately 0.2% of Americans aged 20 to 39 years are childhood cancer survivors.¹ Advances in cancer detection and therapy have greatly improved survival rates for young cancer patients; however, treatment of childhood cancers can adversely impact reproductive function (eg, men who survive childhood cancer are half as likely as their siblings to father a child).² Many cancer patients report a strong desire to be informed of existing options for fertility preservation and future reproduction.³ Therefore, the American Society of Clinical Oncology and the American Society for Reproductive Medicine recommend that consideration of fertility preservation be included prior to initiation of gonadotoxic cancer therapies, including surgery, chemotherapy, and radiotherapy.^4–6Infertility as a result of cancer treatment can be psycho logically upsetting for many patients,^3,7,8 and data suggest that those who pursued fertility preservation usually cope better with their cancer treatment.⁹ Infertile cancer survivors have an option to become parents through adoption or gamete donation, but most declare a preference for having a biological child.^3,10 Schover and colleagues³ found that 51% of newly diagnosed young male cancer patients reported a desire to have children in the future, and this rate increased to 77% for those who did not have children at the time of diagnosis. The desire to become a biological parent persists in male cancer survivors, as 70% reported wanting to father a child after chemotherapy treatment.⁹ A history of cancer treatment may be perceived by some to pose an increased risk to the health of future offspring; however, several studies have shown that male cancer survivors have not demonstrated an increased risk for having a child with birth defects or cancer.^11,12 Recently, a retrospective cohort study conducted in the United States showed no increased risk of malformations or premature birth in the offspring of male cancer survivors.¹³The optimal time for consideration of fertility preservation is before the initiation of any oncologic therapy that can affect gametogenesis; thus, it is critical that fertility preservation is discussed with all patients at the time of diagnosis and before treatment starts. Practitioners who provide care for cancer patients should be aware of the relationship between cancer treatment and infertility. Moreover, they need to be able to appropriately refer patients to a reproductive medicine specialist in a timely fashion for further counseling and fertility preservation. Although fertility concerns are paramount to young adults with cancer, many oncologists still do not routinely address these concerns.^3,14 In a survey of 200 young male cancer survivors who were primarily treated at a comprehensive cancer center, only 51% recalled being offered sperm cryopreservation prior to their cancer treatment.³ Further, it is important to recognize the psychologic stressors associated with a new cancer diagnosis and associated late effects of cancer treatment, such as infertility or early menopause. Findings from several studies support the importance of counseling patients regarding their risk for fertility issues and educating providers regarding the potential fertility preservation options that are available. For example, Babb and colleagues¹⁵ found that, at many institutions, this counseling is already taking place and there is a high rate of discussion with newly diagnosed patients regarding infertility.     

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.     

Vascular Targeted Photodynamic Therapy for Localized Prostate Cancer

Survival for men diagnosed with prostate cancer directly depends on the stage and grade of the disease at diagnosis. Prostate cancer screening has greatly increased the ability to diagnose small and low-grade cancers that are amenable to cure. However, widespread prostate-specific antigen screening exposes many men with low-risk cancers to unnecessary complications associated with treatment for localized disease without any survival advantage. One challenge for urological surgeons is to develop effective treatment options for low-risk disease that are associated with fewer complications. Minimally invasive ablative treatments for localized prostate cancer are under development and may represent a preferred option for men with low-risk disease who want to balance the risks and benefits of treatment. Vascular targeted photodynamic therapy (VTP) is a novel technique that is being developed for treating prostate cancer. Recent advances in photodynamic therapy have led to the development of photosynthesizers that are retained by the vascular system, which provides the opportunity to selectively ablate the prostate with minimal collateral damage to other structures. The rapid clearance of these new agents negates the need to avoid exposure to sunlight for long periods. Presented herein are the rationale and preliminary data for VTP for localized prostate cancer.Key words: Prostate cancer, localized; Minimally invasive ablative treatment for prostate cancer; Photodynamic therapy; WST-09; WST-11; Vascular targeted photodynamic therapy; Padoporfin; Palladium bacteriopheophorbideProstate cancer represents the second most common cause of cancer-related deaths in American men; it is estimated that 27,000 men in the United States died from the disease in 2007.¹ Survival for men with prostate cancer directly depends on the stage and grade of the disease at the time of diagnosis.² These sobering mortality statistics and the more favorable prognosis associated with early detection provide the primary justification for prostate cancer screening, which is performed by measuring the level of serum prostate-specific antigen (PSA) and conducting a digital rectal examination (DRE). It is estimated that 50% of men over the age of 50 years are screened annually for prostate cancer.³Despite widespread acceptance, prostate cancer screening is debated,^4,5 and recommendations for prostate cancer screening are inconsistent. Screening protagonists emphasize that radical prostatectomy increases prostate cancer survival in men with localized disease,⁶ and that the recently observed progressive and significant decline in prostate cancer mortality rates is the direct result of PSA screening and aggressive intervention.⁷ Screening antagonists emphasize the indolent natural history of most prostate cancers detected by screening,⁸ and that the vast majority of men who are treated for prostate cancer do not recognize any survival advantage from early detection and are simply left suffering the ravages of treatment.⁹Both sides of the screening debate have valid arguments. In the absence of widespread screening, many men are denied an opportunity to cure their disease. These men will experience the otherwise preventable consequences of disease progression, which include the development of androgen-insensitive disease¹⁰ and death. However, widespread screening exposes many men to unnecessary complications associated with treatment for localized disease. The challenges are to identify and treat only those cancers that have the biological potential to cause serious and preventable consequences, or to develop treatment options that are associated with fewer complications.     

Effective Testosterone Suppression for Prostate Cancer: Is There a Best Castration Therapy?

Achieving and maintaining effective suppression of serum testosterone levels in men treated with androgen ablation is one of the essential strategies in the management of prostate cancer. Historically, a serum testosterone below 50 ng/dL was considered to be the castrate level. Current data suggest that the new target for either surgical or chemical castration is a serum testosterone level of lower than 20 ng/dL in an attempt to maximize therapeutic outcomes. Testosterone breakthrough and the acute-on-chronic effects of administration of a luteinizing hormone-releasing hormone analogue may cause testosterone levels to periodically rise, sometimes to noncastrate levels. The goal of androgen ablation is to identify those agents that will most consistently achieve and maintain the lowest testosterone levels possible.Key words: Prostate cancer, Androgen ablation, LHRH analogues, LHRH antagonists, TestosteroneThe cornerstone of understanding the basic biology of prostate cancer relies upon the important discovery that prostate cancer is a hormonally responsive tumor. The current use of androgen ablation therapy in prostate cancer includes treatment based on serum prostate-specific antigen (PSA) only or local recurrence; neoadjuvant or adjuvant treatment of high-risk disease, usually in combination with radiation therapy; and treatment of patients with metastatic disease regardless of symptoms. The American Society of Clinical Oncology (ASCO) 2007 guidelines and National Comprehensive Cancer Network (NCCN) 2009 guidelines recommend either luteinizing hormone-releasing hormone (LHRH) agonists or bilateral orchiectomy as first-line therapy for men with advanced prostate cancer.^1,2Medical or chemical castration is almost exclusively performed by the use of injectable LHRH analogues, with a minor role for estrogen and limited experience with LHRH antagonists. Surgical castration through bilateral orchiectomy is infrequently used today.Intermittent hormonal therapy (IHT) is being investigated as an alternative to continuous hormonal therapy with a potential for reduced morbidity and a delay of the progression to hormone-refractory disease.³ Although intermittent therapy may rely upon restoring a normal testosterone level, it is believed that the testosterone level should be as low as possible when the patient is on treatment, thus generating the lowest serum PSA level possible and likely improving outcome.⁴ Although the data on IHT are promising, trials reported thus far are relatively small and somewhat underpowered, and it is likely that its use will increase in the future as trials mature.There is growing recognition that many men may not achieve acceptable levels of testosterone using androgen ablation. This has led to a renewed interest in the significance of the testosterone level in the modern era of prostate cancer management. Can we define the best castration therapy for prostate cancer? Is this the therapy that provides the lowest and most consistent levels of testosterone suppression? To quote Dr. Claude Schulman in a recent editorial: “less is more.”⁵     

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.     

Penile Rehabilitation Strategies Among Prostate Cancer Survivors

Despite advances in technical and surgical approaches, erectile dysfunction (ED) remains the most common complication among prostate cancer survivors, adversely impacting quality of life. This article analyzes the concept and rationale of ED rehabilitation programs in prostate cancer patients. Emphasis is placed on the pathophysiology of ED after diagnosis and treatment of prostate cancer to understand the efficacy of rehabilitation programs in clinical practice. Available evidence shows that ED is a transient complication following prostate biopsy and cancer diagnosis, with no evidence to support rehabilitation programs in these patients. A small increase in ED and in the use of phosphodiesterase type 5 (PDE5) inhibitors was reported in patients under active surveillance. Patients should be advised that active surveillance is unlikely to severely affect erectile function, but clinically significant changes in sexual function are possible. Focal therapy could be an intermediate option for patients demanding treatment/refusing active surveillance and invested in maintaining sexual activity. Unlike radical prostatectomy, there is no support for PDE5 inhibitor use to prevent ED after highly conformal external radiotherapy or low-dose rate brachytherapy. Despite progress in the understanding of the pathophysiologic mechanisms responsible for ED in prostate cancer patients, the success rates of rehabilitation programs remain low in clinical practice. Alternative strategies to prevent ED appear warranted, with attention toward neuromodulation, nerve grafting, nerve preservation, stem cell therapy, investigation of neuroprotective interventions, and further refinements of radiotherapy dosing and delivery methods.Key words: Prostate cancer, Erectile dysfunction, Penile rehabilitation, Phosphodiesterase type 5 inhibitor, Prostaglandin E₁In the United States, prostate cancer is the most frequently diagnosed nonskin cancer in men, and is second only to lung cancer as a cause of cancer death.¹ In 2014, an estimated 233,000 men in the United States were diagnosed with a prostate cancer and 29,480 men were expected to die from their disease.² In the prostate-specific antigen (PSA) era, the importance of this type of cancer becomes evident when considering that more young, sexually active men are being diagnosed at an early stage while the tumor is still organ confined. Early detection of prostate cancer in the PSA era, as well as improvements in systemic treatment of metastatic prostate cancer, has led to an increased life expectancy; but cancer diagnosis and treatment carry serious physical and psychological consequences that can dramatically decrease quality of life.³ However, these results had recently drawn the attention of the scientific community to the quality of life of cancer survivors in order to promote health as defined by the World Health Organization (WHO).⁴ Despite advances in technical and surgical approach, erectile dysfunction (ED) remains the most common and the most documented complication among prostate cancer survivors, adversely impacting quality of life.⁵ ED is also a prevalent long-term complication among prostate cancer patients receiving systemic therapy.⁶ In recent years, investigators have increasingly focused on ED in prostate cancer patients. They have directed their efforts toward searching for interventions that might improve erectile function. Various coping strategies and rehabilitation programs have been suggested and applied with different success rates.This article provides an overview of the literature, analyzing the concept and rationale of rehabilitation programs for ED in prostate cancer patients. Emphasis is placed on the pathophysiology of such disorders after diagnosis and treatment of prostate cancer in order to understand the efficacy of rehabilitation programs in clinical practice.     

The Impact of Prostate Cancer and Hormonal Therapy on Bone

Large-scale studies agree that the observed decline in prostate cancer mortality that began in the early 1990s, shortly after prostate-specific antigen testing was introduced in the United States, is most likely explained by more widespread treatment of prostate cancer, including hormonal therapy. Practitioners should be aware of the risk of the development of osteoporosis and of skeletal side effects related to hormonal therapy to optimize the care of men with prostate cancer.Key words: Hormone deprivation therapy, Androgen deprivation therapy, Bone destruction, Osteoporosis, OsteopeniaProstate cancer is the most commonly diagnosed cancer among men in the United States, with approximately 192,280 cases anticipated in 2009.¹ It also remains a common cause of cancer death, with 27,360 deaths anticipated in 2009. Moreover, the declining US death rates from cardiovascular and smoking-related disease coupled with the aging of the population associated with the Baby Boom generation may beget an anticipated increase in prostate cancer diagnoses in the coming years. It has been estimated that about 10% of the US population was over the age of 65 years in 2000 and that this proportion will approximately double by 2030.² As a condition of aging men, prostate cancer is apt to remain a significant, if not growing, public health problem.Current efforts to reduce the mortality burden of prostate cancer have included prostate-specific antigen (PSA)-based screening, but its effect on mortality as assessed in randomized trials, particularly during the first 10 years of follow-up, is controversial.^3,4 But these large-scale studies agree that the observed decline in prostate cancer mortality that began in the early 1990s, shortly after PSA testing was introduced in the United States, is most likely explained by more widespread treatment of prostate cancer, including hormonal therapy.⁵ Given these considerations, it is quite likely that hormone deprivation therapy will remain an important treatment for men with prostate cancer. Therefore, a thorough understanding of its long-term side effects is necessary if we are to optimize the care of men with prostate cancer.     

Metastatic Urethral Melanoma: Case Report and Review of the Literature

Melanoma is a cancer that originates from melanocytes, is predominant in adults with white skin, represents 4% of skin cancers, and has high possibility of forming metastasis. This review reports on the case of a young man, age 36 years, previously diagnosed with melanoma. The patient complained of obstructive urinary symptoms and, while he was undergoing a cystoscopy, it was discovered that he had a lesion corresponding with metastatic melanoma of the prostatic urethra, which occluded almost the entire urethra and resulted in blocked urinary flow. He underwent a transurethral resection of the prostate, followed by resection of the lesion. After the procedure, he had good urinary flow and is currently on follow-up.Key words: Melanoma, Urethra, Urinary obstruction, Metastasis, Urethral melanomaPrimary malignant melanoma of the urethra is rare, representing < 1% of all melanomas^1,2; it is often misdiagnosed, which leads to delays in treatment.² The lethality is high, but its incidence is low. Prognosis is considered good if it is detected in its early stages.¹ In recent years, there have been great improvements in patient survival rates. In developed countries, the average estimated 5-year survival is 73%, whereas in developing countries, the average survival is 56%. The estimated world average is 69%.¹ Risk factors in order of importance are sensitivity to the sun, light skin, excessive sun exposure, history of skin cancer, family history of melanoma, congenital nevi, maturity, xeroderma pigmentosum, and dysplastic nevi.¹Individual management according to the clinical presentation is based on extrapolation of evidence for other melanoma treatments.² Due to low occurrence rates of urethral melanoma, the optimal therapy has not yet been established, and surgery remains the mainstay of primary therapy; adjuvant locoregional and systemic therapies are needed.² This article reports on the case of a young patient with metastatic melanoma in the urethra which led to urinary obstruction and urinary symptoms.     

Inhibition of Circulating Dipeptidyl Peptidase 4 Activity in Patients with Metastatic Prostate Cancer

Cancer is responsible for many deaths and is a major source of healthcare expenditures. The identification of new, non-invasive biomarkers might allow improvement of the direct diagnostic or prognostic ability of already available tools. Here, we took the innovative approach of interrogating the activity of exopeptidases in the serum of cancer patients with the aim of establishing a distinction based on enzymatic function, instead of simple protein levels, as a means to biomarker discovery. We first analyzed two well-characterized mouse models of prostate cancer, each with a distinct genetic lesion, and established that broad exopeptidase and targeted aminopeptidase activity tests reveal proteolytic changes associated with tumor development. We also describe new peptide-based freeze-frame reagents uniquely suited to probe the altered balance of selected aminopeptidases, as opposed to the full array of exopeptidases, and/or their modulators in patient serum or plasma. One particular proteolytic activity was impaired in animals with aggressive disease relative to cancer-free littermates. We identified the protease in question as dipeptidyl peptidase 4 (DPP4) by analyzing selected knockout mice and evaluating the effect of specific inhibitors. DPP4 activity was also reduced in the sera of patients with metastatic prostate cancer relative to patients with localized disease or healthy controls. However, no significant differences in DPP4 serum levels were observed, which established the loss of activity as the result of impaired enzymatic function. Biochemical analysis indicated that reduced activity was the result not of post-translational modifications or allosteric changes, but instead of a low-molecular-weight inhibitor. After we adjusted for age and total prostate-specific antigen, reduced DPP4 activity remained a significant predictor of cancer status. The results of this proof-of-principle study suggest that DPP4 activity might be a potential blood-based indicator of the presence of metastatic cancer of prostatic origin, either by itself or, more likely, as a means to improve the sensitivity and specificity of existing markers.Biomarkers have featured prominently in tests designed to aid in medical decision making, such as establishing a diagnosis, determining prognosis, and assessing the effects of treatment. In clinical oncology practice, biomarkers are required to address relevant questions related not only to patients with early stage disease, but also to those with metastatic and, in some cases, incurable cancer. An ideal marker for cancer diagnosis and surveillance is one that is noninvasive and reproducible, with high sensitivity and specificity. The classic path to cancer biomarker discovery involves measuring differential levels of proteins in the blood or tissue of interest using immunohistochemical- or mass spectrometry (MS)-based screens. This approach has not been a great success, mainly because the complexity of the blood proteome precludes the detection of proteins and peptides at low levels without time-consuming prefractionation. As a result, disappointingly few assays have been translated into clinical practice so far (1, 2), a regrettable disconnect that advocates conceptually novel biomarker discovery and validation schemes. An example of an alternate approach is examination of the activity of proteins, in particular enzyme families, that are relevant with respect to the disease of interest. In the case of cancer, proteases are one such class, as several of its members have been implicated in promoting both tumor progression and suppression (3–6).It has been suggested that the cumulative exopeptidase activity in blood can provide accurate class discrimination between patients with solid tumors and controls without cancer (7, 8). Initial assessments were made either by carefully measuring and identifying a subset of the endogenous serum peptide metabolome—a notoriously difficult process—or by monitoring the degradation of spiked, synthetic peptide substrates using a method that allows straightforward yet accurate quantitation of the breakdown products on a whole serum proteome background. This method, termed the sequence-specific exopeptidase activity test (SSEAT),¹ provides an aggregate read-out of protease activities and has the important advantage of all but eliminating reproducibility problems related to sample collection, storage, and handling that have beset serum oncopeptidomic studies of the past (8–11). From a classical proteomics point of view, some of these proteases may also be exceedingly low abundant in serum and therefore “invisible” in traditional MS-based discovery schemes. However, given enough substrate, time, and optimal assay conditions, catalytic product may accumulate to such a level that it becomes readily detectable in any type of mass spectrometer. To date, SSEAT assays have never been applied to study well-characterized animal models of cancer to determine whether they may reveal proteolytic changes associated with tumor development or whether such changes are relevant to human cancer.Prostate cancer (PCa) is the most prevalent malignancy in men and the second leading cause of cancer death in North America, with one in six men having a lifetime risk of being diagnosed and a 3.4% chance of death (12). It is a heterogeneous disease, with some patients diagnosed at an early stage who either do not require treatment or are cured following surgery, and some diagnosed with advanced disease or who suffer recurrence despite initial, apparently effective treatment (13, 14). Serum prostate-specific antigen (PSA) is the only protein biomarker routinely used for the detection and management of a common cancer, but it is not a reliable intermediate indicator of overall survival (15–18). As an example, metastatic castration-resistant prostate cancer (mCRPC) is generally associated with poor outcomes, but precise survival times are hard to predict at present (14, 19–21). A newly developed biomarker used independently is unlikely to surpass the accuracy of the current gold standards for diagnosis, but a goal of discovery would be to integrate a new marker in the process of clinical decision making to improve upon the diagnostic or prognostic ability of already existing tools.The current investigation sought to exploit the merits of analyzing mouse models of PCa to establish whether SSEAT assays may reveal proteolytic changes with tumor development and whether such changes are relevant to human disease. We also describe new peptide-based reagents uniquely suited to probe the altered balance of selected aminopeptidases, as opposed to the full array of exopeptidases, and/or their modulators in serum or plasma of cancer patients. Using suitable animal models and individualized assays, we found that DPP4 activity was markedly reduced in serum of mCRPC patients relative to that of patients with localized disease and healthy control individuals. Biochemical analysis suggests the existence of a low-molecular-weight inhibitor of circulating DPP4 that is either uniquely present or at elevated levels in patients with advanced disease. After we adjusted for age and total PSA, DPP4 activity remained a significant predictor of cancer status.     

Chemokine (C-C Motif) Ligand 2 Engages CCR2+ Stromal Cells of Monocytic Origin to Promote Breast Cancer Metastasis to Lung and Bone

Metastatic spread of cancer to distant vital organs, including lung and bone, is the overwhelming cause of breast cancer mortality and morbidity. Effective treatment of systemic metastasis relies on the identification and functional characterization of metastasis mediators to multiple organs. Overexpression of the chemokine (C-C motif) ligand 2 (CCL2) is frequently associated with advanced tumor stage and metastatic relapse in breast cancer. However, the functional mechanism of CCL2 in promoting organ-specific metastasis of breast cancer has not been rigorously investigated. Here, we used organ-specific metastatic sublines of the MDA-MB-231 human breast cancer cell line to demonstrate that overexpression of CCL2 promotes breast cancer metastasis to both lung and bone. Conversely, blocking CCL2 function with a neutralizing antibody reduced lung and bone metastases. The enhancement of lung and bone metastases by CCL2 was associated with increased macrophage infiltration and osteoclast differentiation, respectively. By performing functional assays with primary cells isolated from the wild type, CCL2 and CCR2 knock-out mice, we showed that tumor cell-derived CCL2 depends on its receptor CCR2 (chemokine, CC motif, receptor 2) expressed on stromal cells to exert its function in promoting macrophage recruitment and osteoclast differentiation. Overall, these data demonstrated that CCL2-expressing breast tumor cells engage CCR2⁺ stromal cells of monocytic origin, including macrophages and preosteoclasts, to facilitate colonization in lung and bone. Therefore, CCL2 and CCR2 are promising therapeutic targets for simultaneously inhibiting lung and bone metastasis of breast cancer.Breast cancer is the most common malignancy in women in the United States, with an estimated 182,000 new cases and 40,000 deaths in 2008 (1). Late stage breast cancer patients develop metastases in bone, lung, liver, brain, and other organs, which are responsible for most breast cancer-related mortality and morbidity (2). Severe complications from bone metastasis include debilitating bone fractures, nerve compression and bone pain, and hypercalcemia (3–5), whereas lung metastasis is accompanied by cough, bloody sputum, rib cage pain, and, eventually, failure of the respiratory functions (6). Colonization of different secondary organs by breast cancer is believed to be a complex, multigenic process that depends on productive interactions between tumor cells and stromal microenvironments through concerted actions of organ-specific metastasis genes (7, 8). Functional genomic analysis of preclinical models of breast cancer to bone, lung, and brain have identified distinct sets of organ-specific metastasis genes (9–11), providing novel mechanistic insights into key rate-limiting steps of metastasis to different organs. However, as advanced breast cancer patients often suffer from metastases at several secondary organs, identifying genes that are capable of instigating metastasis to multiple sites may provide the ideal targets for therapeutic intervention of systemic metastasis.Chemokines are small (8–14 kDa) proteins classified into four conserved groups (CXC, CC, C, and CX3C) based on the position of the first two cysteines that are adjacent to the amino terminus (12). They are chemotactic cytokines that stimulate directed migration of leukocytes in response to inflammatory signals. Chemokines are also involved in the maintenance of hematopoietic homeostasis, regulation of cell proliferation, tissue morphogenesis, and angiogenesis (13). Chemokines bind to the seven-transmembrane domain receptors to elicit downstream molecular events that coordinate cell movement. Even though chemokines are unlikely to be a contributing factor for tumor initiation, they can have pleiotropic effects on tumor progression (13, 14). Among more than 50 human chemokines, CCL2 is of particular importance. CCL2, also called monocyte chemoattractant protein-1 (MCP-1), is a potent chemoattractant for monocytes, memory T lymphocytes, and natural killer cells (15). It is involved in a number of inflammatory conditions associated with monocyte recruitment, including delayed hypersensitivity reactions, bacterial infection, arthritis, and renal disease (15). The importance of CCL2 in cancer was manifested by its overexpression in a variety of tumor types, including glioma, ovarian, esophagus, lung, breast, and prostate cancers (15–17). In prostate cancer, CCL2 expression levels was associated with advanced pathological stage (16). Importantly, CCL2-neutralizing antibodies inhibit bone resorption in vitro and bone metastasis in vivo (18–20). In lung cancer, serum CCL2 levels were elevated in lung cancer patients with bone metastasis compared with localized diseases. Neutralizing antibodies against CCL2 also inhibited the tumor conditioned media-induced osteoclast formation in vitro and bone metastasis in vivo (17). Taken together, these findings suggested a role of CCL2 in bone metastasis.A potential role of CCL2 in breast cancer progression and metastasis has been indicated by the analysis of CCL2 expression of tumor and serum samples from breast cancer patients. Serum CCL2 levels were significantly higher in postmenopausal breast cancer patients than in age-matched controls (21). Over 50% of breast cancer tumor samples had intense staining of CCL2 in tumor cells (22). Prognostic analysis further revealed that high expression of CCL2 was correlated with advanced tumor stage, lymph node metastasis (23), and early relapse (24). CCL2 up-regulation in breast tumors was also associated with the infiltration of tissue-associated macrophages (TAMs)³ and with increased microvessel density (22, 24). TAMs have been known to contribute to primary tumor progression and metastasis of breast cancer (25), which is supported by epidemiological evidence showing that TAM infiltration portended a poor clinical outcome (26, 27). However, whether the function of CCL2 in modulating activity of macrophages and possibly other cell types is important for breast tumor organotropic metastasis has not been rigorously investigated. CCL2 may engage organ-specific cell types derived from the same bone marrow myelomonocytic progenitors. These progenitors differentiate into osteoclast precursors in bone or into blood monocytes that eventually become mature macrophages in different tissues, like alveolar macrophages in lung (28). These stromal cell types of myelomonocytic origin may contribute to different functions in different organ-specific metastases. Another unresolved question regarding the function of CCL2 in tumor-stroma interaction is the functional involvement of CCL2 receptors. CCL2 can bind to both CCR2 and CCR4 (29, 30). Loss of function studies in mice showed CCL2 and CCR2 knock-out mice displayed similar impairments in monocyte migration (31, 32), suggesting that CCR2 is the major functional receptor for CCL2. Understanding whether CCR2 deficiency in stromal cells leads to compromised monocyte engagement by CCL2-expressing tumor cells may have important implications in designing targeting therapeutics against the CCL2/CCR2 axis.In this study, we used the recently developed organ-specific metastatic sublines of the human breast cancer cell MDA-MB-231 (9, 10, 33) and showed that overexpression of CCL2 promotes both lung and bone metastases. This function was associated with increased TAM infiltration in lung metastasis and increased osteoclast differentiation in bone metastasis, respectively. Furthermore, by using macrophages and bone marrow cells isolated from wild type, CCL2-deficient, and CCR2-deficient mice, we showed that CCR2 expression in stromal cells is essential for tumor-derived CCL2 to recruit macrophages and promote osteoclastogenesis. Targeting tumor-derived CCL2 by a neutralizing antibody significantly reduced metastasis formation in both bone and lung.