The genomic expression test EndoPredict is a prognostic tool for identifying risk of local recurrence in postmenopausal endocrine receptor-positive, her2neu-negative breast cancer patients randomised within the prospective ABCSG 8 trial.

BACKGROUND: The aim of this study was to examine whether EndoPredict (EP), a novel genomic expression test, is effective in predicting local recurrence (LR)-free survival (LRFS) following surgery for breast cancer in postmenopausal women. In addition, we examined whether EP may help tailor local therapy in these patients. METHODS: From January 1996 to June 2004, 3714 postmenopausal patients were randomly assigned to either tamoxifen or tamoxifen followed by anastrozole within the prospective ABCSG 8 trial. Using assay scores from EP, we classified breast tumour blocks as either low or high risk for recurrence. RESULTS: Data were gathered from 1324 patients. The median follow-up was 72.3 months and the cumulative incidence of LR was 2.6% (0.4% per year). The risk of LR over a 10-year period among patients with high-risk lesions (n=683) was significantly higher (LRFS=91%) when compared with patients with low-risk lesions (n=641) (10-year LRFS=97.5%) (HR: 1.31 (1.16-1.48) P<0.005). The groups that received breast conservation surgery (BCT) and mastectomy (MX) had similar LR rates (P=0.879). Radiotherapy (RT) after BCT significantly improved LRFS in the cohorts predicted by EP to be low-risk for LR (received RT: n=436, 10-year LRFS 99.8%; did not receive RT: n=63, 10-year LRFS 83.6%, P<0.005). CONCLUSIONS: EndoPredict is an effective prognostic tool for predicting LRFS. Among postmenopausal, low-risk patients, EP does not appear to be useful for tailoring local therapy.

The EndoPredict score provides prognostic information on late distant metastases in ER+/HER2- breast cancer patients.

BACKGROUND: ER+/HER2- breast cancers have a proclivity for late recurrence. A personalised estimate of relapse risk after 5 years of endocrine treatment can improve patient selection for extended hormonal therapy. METHODS: A total of 1702 postmenopausal ER+/HER2- breast cancer patients from two adjuvant phase III trials (ABCSG6, ABCSG8) treated with 5 years of endocrine therapy participated in this study. The multigene test EndoPredict (EP) and the EPclin score (which combines EP with tumour size and nodal status) were predefined in independent training cohorts. All patients were retrospectively assigned to risk categories based on gene expression and on clinical parameters. The primary end point was distant metastasis (DM). Kaplan-Meier method and Cox regression analysis were used in an early (0-5 years) and late time interval (>5 years post diagnosis). RESULTS: EP is a significant, independent, prognostic parameter in the early and late time interval. The expression levels of proliferative and ER signalling genes contribute differentially to the underlying biology of early and late DM. The EPclin stratified 64% of patients at risk after 5 years into a low-risk subgroup with an absolute 1.8% of late DM at 10 years of follow-up. CONCLUSION: The EP test provides additional prognostic information for the identification of early and late DM beyond what can be achieved by combining the commonly used clinical parameters. The EPclin reliably identified a subgroup of patients who have an excellent long-term prognosis after 5 years of endocrine therapy. The side effects of extended therapy should be weighed against this projected outcome.

EndoPredict improves the prognostic classification derived from common clinical guidelines in ER-positive, HER2-negative early breast cancer.

BACKGROUND: In early estrogen receptor (ER)-positive/HER2-negative breast cancer, the decision to administer chemotherapy is largely based on prognostic criteria. The combined molecular/clinical EndoPredict test (EPclin) has been validated to accurately assess prognosis in this population. In this study, the clinical relevance of EPclin in relation to well-established clinical guidelines is assessed. PATIENTS AND METHODS: We assigned risk groups to 1702 ER-positive/HER2-negative postmenopausal women from two large phase III trials treated only with endocrine therapy. Prognosis was assigned according to National Comprehensive Cancer Center Network-, German S3-, St Gallen guidelines and the EPclin. Prognostic groups were compared using the Kaplan-Meier survival analysis. RESULTS: After 10 years, absolute risk reductions (ARR) between the high- and low-risk groups ranged from 6.9% to 11.2% if assigned according to guidelines. It was at 18.7% for EPclin. EPclin reassigned 58%-61% of women classified as high-/intermediate-risk (according to clinical guidelines) to low risk. Women reclassified to low risk showed a 5% rate of distant metastasis at 10 years. CONCLUSION: The EPclin score is able to predict favorable prognosis in a majority of patients that clinical guidelines would assign to intermediate or high risk. EPclin may reduce the indications for chemotherapy in ER-positive postmenopausal women with a limited number of clinical risk factors.

Four years experience with teleneuropathology.

Between 2002 and 2005, we made 343 intraoperative frozen section diagnoses with a telepathology system, which connected a neurosurgical department to our department of pathology. An expert neuropathologist performed at least one brief gross examination, and this was followed by a smear preparation and a frozen section slide for each case. Frozen section diagnosis lasted on average 26.1 min, calculated from the beginning of gross examination until the surgeon was given the diagnosis. The majority of cases (283 or 83%) were diagnosed in 15-40 min. The mean time needed for macroscopic examination was 3.0 min, time for staining 4.2 min, smear diagnosis took 5.4 min and time for histological diagnosis 10.7 min. Telemicroscopy of a smear slide took 11 times longer compared with light microscopy, and telemicroscopy of a frozen section slide took 16 times longer than with light microscopy. In 6% of cases, the telepathology software posed technical problems, which delayed the time of diagnosis, but not by more than 4 min. We were able to render a diagnosis in all cases (system reliability 100%). After eliminating sampling errors (i.e. cases with no diagnostic material in the frozen section slides and/or in smear preparations), the diagnostic accuracy for telepathology was 97.9%.

Electron Microscopical Autometallography: Immunogold-Silver Staining (IGSS) and Heavy-Metal Histochemistry

Immunogold-silver staining (IGSS) utilizes a histochemical method called autometallography (AMG) to amplify tiny gold particles to sizes easily visible both in light and electron microscopy. In both applications it is advisable to use the smallest possible gold diameters (1-6 nm) to obtain the highest sensitivity, thus, allowing minute amounts of the target substance to be demonstrated. Gold labels smaller than 10 nm in diameter have been clearly shown to give the highest labeling densities of antigen-antibody binding sites. AMG can be used for the detection of catalytic crystal lattices of metallic gold and silver, and sulfides or selenides of mercury, silver, copper, bismuth, and zinc. The method has its roots in "physical development" technique, transplanted from photography to histology by Liesegang at the beginning of this century. In 1981, a series of papers were published by one of us with the purpose of introducing a reliable and easy-to-handle technique for light microscopical and ultrastructural studies. AMG has a multitude of applications apart from its use in detecting tissue metals. These include the highly sensitive and efficient in situ colloidal gold tracing of peptides, proteins, and amines by immunocytochemistry using the IGSS method, of carbohydrates by lectin IGSS, and of nucleic acids by IGSS in situ hybridization, IGSS in situ polymerase chain reaction, and IGSS in situ self-sustained sequence replication-based amplification (in situ 3SR) techniques, the last two even performing with single-copy sensitivity. Applications of pre- and postembedding AMG for semithin and ultrathin tissue sections are described.

Monoclonal antibody B-ly7: a sensitive marker for detection of minimal residual disease in hairy cell leukemia.

The new monoclonal antibody (MoAb) B-ly7 was tested for its value in bone marrow diagnosis in patients with hairy cell leukemia (HCL). Cryostat sections of bone marrow biopsies were examined by an indirect immunoperoxidase technique. Lymphoma cells from all of 26 HCL cases investigated displayed strong surface membrane staining with the MoAb B-ly7, whereas tumor cells from only one of 63 patients with other lymphoproliferative disorders of B cell type reacted with this antibody. The strong reactivity of hairy cells (HCs) with this marker was not altered after therapy as demonstrated on control biopsies taken from patients treated with interferon(IFN)-alpha-2 or 2'deoxycoformycin(DCF) six-64 weeks after start of treatment. This fact as well as the very low number of B-ly7 positive cells found in a series of 13 normal bone marrow biopsies (mean: 0.3% of bone marrow cells, range: 0.0%-1.0%), which could easily be distinguished from HCs by their lower staining intensity and their morphological appearance, provided the basis for the detection of even single HCs. In our hands, in terms of sensitivity the immunohistological detection of HCs using the MoAb B-ly7 was not only superior to classical morphological techniques but also to other immunohistological parameters usually applied for this purpose. Therefore, this MoAb provides a marker for the identification of HCs, hence monitoring disease activity in HCL, and particularly for a critical response evaluation in patients undergoing treatment with IFN-alpha or DCF.

Morphology of acute rejection and corresponding cytological findings in exocrine secretion after pancreas transplantation in the rat.

Reliable and timely rejection diagnosis still represents a major problem of pancreas allotransplantation. The aim of this study was to confirm the clinical findings of exocrine function impairment and pancreatic juice cytology during rejection, to refine the latter by means of flow cytometry, and to correlate these changes with graft histology. Heterotopic pancreatic transplants were performed in a modified technique in Lewis rats rendered diabetic by means of streptozotocin from LEW donors (group I, n = 10), Brown Norway rats without immunosuppression (group II, n = 16), and from BN rats where recipients were given cyclosporine 12 mg/kg/BW (group III, n = 10). Pancreatic juice was obtained by daily aspiration from a self-made fully implantable catheter reservoir system. In group II animals acute rejection diagnosed on histomorphological grounds was clearly associated with a decrease in the amount of exocrine secretion and its enzyme content from day 8 on. In contrast to groups I and III, a significant increase in lymphocytes in the pancreatic juice up to 13.5% occurred in group II between days 5 and 7. Activated lymphocytes increased from 7% to 13%, pan-T cells from 193 to 340 events. Histology revealed three distinct phases of acute rejection--phase I: diffuse infiltration of acinar structures; phase II: destruction of interlobular ducts; phase III: vasculitis associated with islet cell damage. The anatomy of the pancreas with the slackness of its highly vascularized interstitial connective tissue facilitates early infiltration of inflammatory cells and migration of these cells into the lumen of the pancreatic duct. Thus pancreatic juice cytology together with an impaired exocrine graft function is highly indicative of acute rejection.

At what stage does pancreas allograft rejection become irreversible?: an experimental study.

BACKGROUND: It is commonly believed that abnormal blood glucose levels indicate irreversible rejection. We were interested in determining the stage at which rejection remains reversible. METHODS: A total of 54 Lewis rats were rendered diabetic with 55 mg/kg streptozocin and were then given a pancreas transplant from Brown Norway donors. Pancreatic juice was collected in a subcutaneous reservoir. All recipients received 15 mg/kg cyclosporine A (CsA) for 5 days. CsA was then discontinued for 2 days (n=7, group 1), 4 days (n=7, group 2), 6 days (n=9, group 3), 8 days (n=9, group 4), 9 days (n=11, group 5), and 10 days (n=11, group 6). Two animals of each group were euthanized at the end of the immunosuppressive-free interval, for histological assessment of the grade of rejection (G0, GI, GII, GIII). Rejection was treated with methylprednisolone (7 mg/kg body weight) and CsA (15 mg/kg body weight). The volume of pancreatic juice, together with juice cytology (C0, CI, CII) and blood glucose levels, was assessed daily. RESULTS: Blood glucose remained normal throughout the observation period in animals with GI and GII rejection. The numbers of animals that became diabetic were as follows: 5 of 9 (group 4), 7 of 11 (group 5), and 8 of 11 (group 6). Decreased amounts of pancreatic juice were observed in all animals, except those in group 1. The histology returned to normal after anti-rejection therapy in four animals (57%) of group 1, in two animals (28%) of group 2, and in one animal (11%) of groups 3 and 4, respectively. Although there was no animal in groups 5 and 6 with normal graft histology after treatment, there were still four (36%) and three (27%) animals, respectively, that were normoglycemic and that had pancreatic grafts with well-preserved islets. CONCLUSIONS: From these data, we conclude that even GIII rejection with severe endothelialitis and isleitis can be reversed. Therefore, we suggest that a trial of enhanced immunosuppression is justified in patients with advanced pancreas allograft rejection.

Neopterin excretion after liver transplantation and its value in differential diagnosis of complications.

The aim of this study was to investigate patterns and clinical utility of a neopterin marker in liver allograft recipients. Urinary neopterin levels were studied in 59 transplant patients daily until discharge. During the early postoperative period (days +1 to +20) neopterin excretion exhibited a liver-specific pattern that clearly differed from that seen in renal and bone marrow transplant recipients. Neopterin concentrations increased and reached peak values on day +7. The height of this early peak varied in context with complications such as infection or rejection, and was correlated with an unfavorable prognosis. In contrast to the early phase, no liver-specific pattern was observed after day 20. As seen in all other transplant patient populations, values normalized in patients with an uncomplicated course but again increased during infection or rejection episodes. Neopterin levels were particularly high during CMV infection, and their increments were directly related to the severity of CMV disease. The neopterin marker, although not specific, enables discrimination between patients with a complicated and uncomplicated clinical course. High neopterin values early after transplantation are associated with unfavorable prognosis and correlate with an increased risk of developing CMV disease.

Localization and distribution of vasoactive neuropeptides in the human placenta.

Neuropeptides play an important role in the regional regulation of blood flow and hormone secretion. Few studies report the presence of peptides in the human placenta. Our experiment evaluates neuropeptides in the human placenta using immunocytochemical techniques. Representative tissue sections from full-term placentae were fixed immediately after delivery and processed into paraffin sections or frozen. They were treated with multiple immunofluorescence, streptavidin-biotin-peroxidase complex and immunogold-silver staining techniques in combination with well-established monoclonal and polyclonal antibodies, using appropriate absorption controls to ensure the validity of the staining. Vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), neuropeptide tyrosine (NPY), galanin, somatostatin, met-enkephaline, helodermin and substance P-like immunoreactivities were demonstrated within decidual cells. Endothelin-1 was found in both trophoblasts and endothelial cells. Peptide immunoreactivities in the human placenta especially at the decidual interface between mother and fetus supports a role for the diffuse neuroendocrine system (DNES) in the regulation of placental blood flow critical for fetal growth and development.

Immunohistological assessment of bone marrow biopsies from patients with hairy cell leukemia: changes following treatment with alpha-2-interferon and deoxycoformycin.

Forty-six bone marrow biopsies from twelve hairy cell leukemia (HCL) patients, treated with either interferon(IFN)-alpha-2 (n = 8) or 2'deoxycoformycin(DCF) (n = 4), were examined using cryostat sections and an immunoperoxidase technique. Using this sensitive method we were able to demonstrate residual hairy cell (HC) infiltration in five cases, in which evaluation with conventional staining techniques on plastic embedded biopsies revealed complete remission. The amount of HCs in these five samples ranged from 1 to 7% (mean: 3%) of bone marrow cells. Consecutive biopsies in individual HCL patients revealed no changes of the immunological phenotype (CD19, CD22, CD25, CD10, CD11c, FMC7, HLA-DR, surface immunoglobulins) during IFN and DCF treatment. Within the infiltrated bone marrow a considerable number of "reactive" T lymphocytes was identified with prevalence of the T-helper (CD4+) subtype in untreated cases, whereas T-suppressor/cytotoxic (CD8+) cells were within the normal range. IFN treatment resulted in a reduction of CD4+ T lymphocytes (p less than 0.02). Minor alterations of CD8+ T lymphocytes and NK cells (HNK-1 + lymphoid cells) were found in bone marrow during IFN treatment. In DCF-treated patients bone marrow T lymphocytes were markedly reduced below the values of normal bone marrow. This DCF-induced T-cell depression might be related to the clinical observation of persistent cellular immune dysfunctions in HCL patients despite a DCF-induced remission.

Concomitant changes of messenger ribonucleic acid levels of secretogranin II, VGF, vasopressin and oxytocin in the paraventricular nucleus of rats after adrenalectomy and during lactation.

In situ hybridization was used to study the mRNA levels for secretogranin II and VGF in comparison with those of oxytocin and vasopressin in the hypothalamus of rats. VGF is a widespread constituent of large dense core vesicles which is selectively induced in PC12 cells by nerve growth factor. After adrenalectomy the mRNA levels of secretogranin II, VGF and vasopressin were increased 4- to 5-fold in the parvocellular neurons of the paraventricular nuclei. In lactating rats the message for oxytocin and secretogranin II were significantly elevated in the magnocellular neurons of the paraventricular and supraoptic nuclei, whereas for VGF only a smaller non-significant increase was observed. As shown by immunoelectron microscopy secretoneurin (a peptide derived from secretogranin II) and oxytocin are co-stored in the large dense core vesicles of the hypothalamo-neurohypophysial neurons. These results demonstrate that stimulation of both parvo- and magnocellular neurons of the hypothalamus induces a concomitant increase of the messages for secretogranin II and VGF together with those of vasopressin and oxytocin.

Detection of monoclonal B-cell populations in decalcified, plastic-embedded bone marrow biopsies with the polymerase chain reaction.

Polymerase chain reaction (PCR) has been employed successfully for the detection of clonal immunoglobulin gene rearrangements in paraffin-embedded clinical samples. The authors examined whether this technique can also be applied to fixed, decalcified, and plastic-embedded bone marrow biopsies. DNA extracted from 66 glycolmethacrylate-embedded trephine biopsy samples was amplified for the detection of rearranged VDJ regions of the immunoglobulin heavy chain genes using both a single-step and a semi-nested PCR technique. After exclusion of samples with inadequate DNA, clonality was confirmed in 16 (67%) of 24 cases with B cell malignancy, whereas all 11 non-B cell neoplasms, and 6 of 9 cases with normal bone marrow showed evidence of a polyclonal B cell population. Patterns indicating oligo- or monoclonality were observed in three plastic-embedded samples of normal bone marrow, although control PCR of frozen bone marrow samples obtained in parallel showed no evidence of clonality. Repeated PCR of these cases revealed inconsistent bands, probably due to the amplification of rare templates from polyclonal B cells. Decalcified, plastic-embedded bone marrow biopsies are suitable for PCR-based determination of B-cell clonality. To exclude the possibility of false-positive results, monitoring of template DNA quality and independent control amplifications are mandatory.

Hepatocellular transplantation into the lung in chronic liver failure following bile duct obstruction in the rat.

Injection of hepatocytes or cell-free supernatant into the lung was able to prevent death from surgically induced fulminant hepatic failure in the rat in over 90% and 53% of subjects, respectively. The aim of this study was to investigate whether this technique can be applied in chronic liver failure. Chronic liver failure was induced in Lewis rats by ligation and transection of the common bile duct, which led to cirrhosis after 3-5 wk in all animals. Four groups of animals were formed: group 1 (n = 5), normal rats, serving as control; group 2 (n = 15), cirrhotic rats, no further treatment; group 3 (n = 14), hepatocyte transplantation by injection of cell suspension transcutaneously into the right lung of cirrhotic animals four wk after bile duct ligation; group 4 (n = 17), injection of 1 mL cell-free supernatant intravenously at two-day intervals, starting 4 wk after ligation. Liver function tests, prothrombin time and serum protein levels were measured weekly before and every two days after transplantation. In group 2 all animals had died 56 (49-69) days after ligation. Survival in groups 3 and 4 was similar: all rats had died from liver failure 61 (51-72) and 60 (49-76) days following bile duct ligation. Survival rates and laboratory investigations showed no significant differences between treated and untreated cirrhotic animals. These data suggest that hepatocyte transplantation into the lung as well as supernatant injection do not have any significant effect on chronic hepatic failure, at least in the rat model.

Peptidergic innervation of the human clitoris.

In the present study, the distributions of neuropeptides in the normal human clitoris and in a clitoris from an adrenogenital syndrome (AGS) was demonstrated by immunohistochemistry (IHC). Immunohistochemical screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), neuropeptide tyrosine (neuropeptide Y), C-flanking peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP) and substance P immunoreactivities. Special attention was given to the VIP-related peptide helospectin, that has been detected in neuronal elements in the clitoris. No visible differences between the localization and distribution of peptidergic nerve fibers of normal and hypertrophic clitoris from AGS have been observed. Co-localization studies showed the co-existence of VIP, PHM and partly helospectin and neuropeptide Y with CPON within nerve fibers in the cavernous tissue and substance P and CGRP co-expression in nerve fibers especially underneath and within the glans clitoris.

CGRP and substance P in intraepithelial neuronal structures of the human upper respiratory system.

The distribution of intraepithelial nerve fibres and neuroendocrine cells within the surface and glandular epithelium of human nasal mucosa and larynx was examined using immunohistochemical techniques. Neuronal structures were immunostained for the general neuroendocrine marker protein gene-product (PGP) 9.5, and the two neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) using immunofluorescence and streptavidin-biotin-peroxidase complex (S-ABC) methods. Intraepithelial nerve fibres with free nerve endings contained PGP 9.5 and were found within the respiratory surface epithelium of the nasal mucosa and the squamous epithelium of the larynx. A subpopulation of these nerve fibres showed positive immunoreactivties with antibodies against SP and CGRP. Nerve fibres within the ductal epithelium of subepithelial excretory ducts passing the basal membrane and reaching the luminal part were detected. These nerve fibres showed CGRP-like immunoreactivity but not for SP. A dense network of nerve fibres within the squamous surface epithelium was detected in the subglottic and epiglottic region containing CGRP and SP in a small subpopulation of nerve fibres. Single intraepithelial taste buds in the epiglottic region and neuroendocrine cells within the subglottic epithelium expressed PGP 9.5.

Helospectin and pituitary adenylate cyclase activating polypeptide in the human vagina.

Helospectin and pituitary adenylate cyclase activating polypeptide (PACAP), both recently isolated from the poisonous saliva of the American lizard or from ovine hypothalamus respectively, belong to the same peptide family as vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM) and glucagon. In the present study, occurrence and distribution patterns of nerve fibers containing helospectin- and PACAP-like immunoreactivity in the human vagina were investigated by immunohistochemistry. Double immunofluorescent labeling showed that helospectin or PACAP are co-expressed with VIP and PHM within subpopulations of VIP-immunoreactive nerve fibers. Nervous structures containing helospectin and VIP were particularly numerous in the internal mucous lining of the vagina and in free epithelial nerve endings, and an abundant network of nerve fibers surrounding blood vessels was detected. Nerve fibers co-expressing PACAP and VIP were more numerous than those expressing helospectin and VIP and were mainly found in close association with blood vessels as well as beneath and within the epithelium. Due to the lack of non-rabbit helospectin or PACAP antibodies, possible co-localizations between these two peptides could not be investigated at this time. The localizations demonstrated suggest possible roles of the two peptides in the regulation of local blood flow and lubrication of the vagina.

Distribution of two VIP-related peptides, helospectin and pituitary adenylate cyclase activating peptide (PACAP), in the human upper respiratory system.

Helospectin (HS) and pituitary adenylate cyclase activating peptide (PACAP) are newly discovered peptides isolated from the salivary gland venom of the lizard Heloderma horridum and the ovine hypothalamus, respectively. They show chemical similarities to vasoactive intestinal polypeptide (VIP), appear to have similar functions and are present in gut, brain, lung, male and female genitourinary tract. In the present study, the distribution of the helospectin and PACAP-27 in the human upper respiratory system was investigated using indirect immunofluorescence and electron-microscopical ABC-pre-embedding methods. Immunohistochemistry revealed helospectin-like (HS-LI) and PACAP-like (PACAP-LI) immunoreactivity in nerve fibers in human nasal, the larynx (vocal cord, ventricular fold, epiglottis), the tongue and the soft palate mucosa. Helospectin-LI and PACAP-LI containing nerve fibers were mainly found in close association to blood vessels and glandular structures. Colocalization studies carried out by application of double immunofluorescence showed that HS and/(or) PACAP-LI coexist with VIP in apparently the same nerve fibers in the upper respiratory system, although single nerve fibers seem to exclusively express helospectin. The localization patterns of helospectin and PACAP-LI in the human upper respiratory system suggests their possible involvement in the regulation of secretory activities and local blood flow.

Neuropeptides in the human penis: an immunohistochemical study.

In the present study, the distribution of neuropeptides in the human penis is demonstrated by immunohistochemistry (IHC). IHC screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine-methionine (PHM), prepro-VIP (111-122), neuropeptide Y (NPY), C-flanking peptide of NPY (C-PON), calcitonin gene-related peptide, substance P, and galanin immunoreactivities. Special attention was also given to the recently isolated, VIP-related lizard peptide helospectin, which could also be detected in neuronal elements in the penis. Colocalization studies showed the coexistence of VIP, PHM, and partly helospectin, and of NPY with C-PON within nerve fibers in the cavernous and spongious body, the glans penis, and the urethra.