MicroRNA expression profiles of seminoma from paraffin-embedded formalin-fixed tissue.

In this study, we used microRNA (miRNA) microarrays in an unbiased screen for aberrantly expressed miRNAs in seminoma, a primitive type of germ cell tumor. Formalin-fixed and paraffin-embedded (FFPE) surgical samples from 11 cases of normal testicular tissue resected for nonneoplastic causes and from 11 cases of seminoma were assessed for miRNA expression. Normal testicular tissue and seminoma were paired by race. We found 112 miRNAs to be differentially expressed between seminoma and normal testicular tissue; 52 miRNAs were overexpressed, and 60, downregulated in seminoma. We did not observe significant differences between black and white populations in our race-paired study. The upregulation of the expression of hsa-mir-21, hsa-mir-372, hsa-mir-373, has-mir-221, and hsa-mir-222 was validated by reverse transcription and real-time PCR. Hsa-mir-372 was upregulated around 1,270-fold (95 % confidence interval (CI) 525.2-3,064.8; p = 8.1e-5 by Mann-Whitney U test). Hsa-mir-373 was upregulated around 1,530-fold (95 % CI 620.5-3,785.6; p = 8.0e-5 by Mann-Whitney U test), consistent with previous reports, indicating that the miRNAs in FFPE are well preserved, and FFPE can be a valuable source for the miRNA study of seminoma. In addition, expression of hsa-mir-21 (12.2-fold, 0.0095), hsa-mir-221 (3.8-fold, 0.014) and hsa-mir-222 (3.8-fold, 0.019) was found elevated in seminoma compared to normal testicular tissue.

Association of HLA polymorphisms with post-transplant lymphoproliferative disorder in solid-organ transplant recipients.

The association between HLA polymorphisms and PTLD was investigated in a case-control study, comparing 110 predominantly adult solid-organ transplant recipients who developed PTLD to 5601 who did not. Donor and recipient HLA were analyzed. We detected a significant association between recipient HLA-A26 and the development of PTLD (OR 2.74; p = 0.0007). In Caucasian recipients, both recipient and donor HLA-A26 were independently associated with development of PTLD (recipient A26 OR 2.99; p = 0.0004, donor A26 OR 2.81; p = 0.002). Analysis of HLA-A and -B haplotypes revealed that recipient HLA-A26, B38 haplotype was strongly correlated with a higher incidence of EBV-positive PTLD (OR 3.99; p = 0.001). The common ancestral haplotype HLA-A1, B8, DR3, when carried by the donor, was protective against PTLD (OR 0.41; p = 0.05). Several other HLA specificities demonstrated associations with clinical and pathological characteristics as well as survival. These findings demonstrate the importance of HLA polymorphisms in modulating the risk for PTLD, and may be useful in risk stratification and development of monitoring and prophylaxis strategies.

Resolution of nephrotic syndrome after successful bariatric surgery in patient with biopsy-proven FSGS.

The incidence of obesity-related nephropathy (ORG) is increasing with the growing incidence of obesity. ORG is associated with morbid obesity, proteinuria and renal biopsy findings of focal global and segmental glomerulosclerosis (FSGS), which can be associated with significant renal impairment. Weight reduction is associated with improvement of ORG, however, conservative measures aiming at long-term weight reduction are difficult to achieve. Bariatric surgery is the most effective way of achieving long-term weight reduction. We present a case of ORG with nephrotic-range proteinuria and FSGS on renal biopsy. Following bariatric surgery, patient achieved successful weight reduction with significant decrease in proteinuria and stabilization of renal function.

Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization and motility of breast cancer cells.

Prolactin (PRL) stimulates the cytoskeletal re-organization and motility of breast cancer cells. During PRL receptor signaling, Vav2 becomes phosphorylated and activated, an event regulated by the serine/threonine kinase Nek3. Given the regulatory role of Vav2, the function of Nek3 in PRL-mediated motility and invasion was examined. Overexpression of Nek3 in Chinese hamster ovary transfectants potentiated cytoskeletal re-organization in response to PRL. In contrast, downregulation of Nek3 expression by small-interfering RNA (siRNA) attenuated PRL-mediated cytoskeletal reorganization, activation of GTPase Rac1, cell migration and invasion of T47D cells. In addition, PRL stimulation induced an interaction between Nek3 and paxillin and significantly increased paxillin serine phosphorylation, whereas Nek3 siRNA-transfected cells showed a marked reduction in paxillin phosphorylation. Analysis of breast tissue microarrays also demonstrated a significant up-regulation of Nek3 expression in malignant versus normal specimens. These data suggest that Nek3 contributes to PRL-mediated breast cancer motility through mechanisms involving Rac1 activation and paxillin phosphorylation.

Stabilization of prolactin receptor in breast cancer cells.

The role of the hormone prolactin (PRL) in the pathogenesis of breast cancer is mediated by its cognate receptor (PRLr). Ubiquitin-dependent degradation of the PRLr that negatively regulates PRL signaling is triggered by PRL-mediated phosphorylation of PRLr on Ser349 followed by the recruitment of the beta-transducin repeats-containing protein (beta-TrCP) ubiquitin-protein isopeptide ligase. We report here for the first time that interaction between PRLr and beta-TrCP is less efficient in human breast cancer cells than in non-tumorigenic human mammary epithelial cells. Furthermore, we demonstrate that both PRLr degradation and PRLr phosphorylation on Ser349 are impaired in breast tumor cells and tissues, an observation that directly correlates with enhanced expression of the PRLr in malignant breast epithelium. These findings represent a novel mechanism through which altered PRLr stability may directly influence the pathogenesis of breast cancer.

Annexin-V imaging for noninvasive detection of cardiac allograft rejection.

Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.

Successful reversal of severe refractory cardiac allograft rejection by photopheresis.

We treated 4 patients with refractory International Society of Heart and Lung Transplantation Grades IIIA to IV cardiac allograft rejection with extracorporeal photopheresis. Following treatment on 2 consecutive days, 3 patients demonstrated complete histologic reversal of rejection. The remaining patient improved more gradually, but manifested complete cessation of rejection following three 2-day treatments. We conclude that photopheresis is a safe and effective modality for the treatment of severe refractory cardiac allograft rejection and that these results support the use of photopheresis in this clinical setting.

Predicting prostate specific antigen outcome preoperatively in the prostate specific antigen era.

PURPOSE: We evaluated the ability of previously defined risk groups to predict prostate specific antigen (PSA) outcome 10 years after radical prostatectomy in patients diagnosed with clinically localized prostate cancer during the PSA era. MATERIALS AND METHODS: Between 1989 and 2000, 2,127 men with clinically localized prostate cancer underwent radical prostatectomy, including 1,027 at Hospital of the University of Pennsylvania (study cohort) and 1,100 at Brigham and Women's Hospital (validation cohort). Cox regression analysis was done to calculate the relative risk of PSA failure with the 95% confidence interval (CI) in patients at intermediate and high versus low risk. The Kaplan-Meier actuarial method was used to estimate PSA outcome 10 years after radical prostatectomy. RESULTS: Compared with low risk patients (stages T1c to 2a disease, PSA 10 ng./ml. or less and Gleason score 6 or less) the relative risk of PSA failure in those at intermediate (stage T2b disease or PSA greater than 10 to 20 ng./ml. or less, or Gleason score 7) and high (stage T2c disease, or PSA greater than 20 ng./ml. or Gleason score 8 or greater) risk was 3.8 (95% CI 2.6 to 5.7) and 9.6 (95% CI 6.6 to 13.9) in the study cohort, and 3.3 (95% CI 2.3 to 4.8) and 6.3 (95% CI 4.3 to 9.4) in the validation cohort. The 10-year PSA failure-free survival rate in the 1,020 patients in the low, 693 in the intermediate and 414 in the high risk groups was 83%, 46% and 29%, respectively (p <0.0001). CONCLUSIONS: Based on 10-year actuarial estimates of PSA outcome after radical prostatectomy 3 groups of patients were identified using preoperative PSA, biopsy Gleason score and 1992 clinical T category.

Estimating the impact on prostate cancer mortality of incorporating prostate-specific antigen testing into screening.

OBJECTIVES: Whether early detection using prostate-specific antigen (PSA) and digital rectal examination (DRE) compared with DRE alone will reduce prostate cancer mortality awaits the results of ongoing prospective randomized trials. However, the impact that early detection could have on prostate cancer-specific survival can be estimated by assuming that PSA failure after radical prostatectomy (RP) will translate into death from prostate cancer. METHODS: The study population consisted of 1274 men with clinically localized prostate cancer who underwent RP in Boston, Massachusetts or Philadelphia, Pennsylvania between 1989 and 2000 and had a preoperative PSA level greater than 4 but not more than 10 ng/mL. The primary endpoint was actuarial freedom from PSA failure (defined as PSA outcome). RESULTS: The relative risk of PSA failure after RP for patients diagnosed with a PSA of greater than 4 to 5, 5 to 6, 6 to 7, or 7 to 8 ng/mL compared with greater than 8 up to 10 ng/mL was 0.3 (95% confidence interval [CI] 0.2 to 0.5), 0.5 (95% CI 0.4 to 0.8), 0.6 (95% CI 0.4 to 0.9), or 0.9 (95% CI 0.6 to 1.3), respectively. On the basis of the estimates of the 5-year PSA outcome, patients with a biopsy Gleason score of 5 or 6 (781 of 1274; 61%) consistently benefited from RP performed when the PSA at diagnosis was greater than 4 to 7 ng/mL compared with greater than 8 to 10 ng/mL (93% versus 78%, P <0.0001). A benefit to early detection was not found for the vast majority (266 of 312; 88%) of patients who had a biopsy Gleason score of 7 or higher. CONCLUSIONS: Early detection using both PSA and DRE-based screening may benefit men who present with biopsy Gleason score 5 or 6 prostate cancer and a PSA level greater than 4 to 7 ng/mL compared with greater than 8 up to 10 ng/mL. This finding awaits validation from ongoing prospective randomized trials.

Metachronous Epstein-Barr virus-related smooth muscle tumors in a child after heart transplantation: case report and review of the literature.

Soft tissue tumors are uncommon manifestations of Epstein-Barr virus (EBV) infection in patients who have had transplants. The authors report 2 metachronous EBV-containing smooth muscle tumors in a child who had a heart transplant, and review the literature on posttransplant soft tissue tumors.

Emergent lung retransplantation after discovery of two primary malignancies in the donor.

A 50-year-old woman underwent single lung transplantation for advanced chronic obstructive pulmonary disease. Shortly after the procedure, it was discovered that the donor suffered from both a renal cell carcinoma and a spindle-cell sarcoma of the ascending aorta, which had metastasized to the spleen. The patient was emergently listed for a retransplantation and underwent bilateral lung transplantation after a new donor became available 4 days after the initial transplantation procedure. After 24 months, the patient is without evidence of malignancy. This case illustrates the role of immediate retransplantation for patients who have inadvertently received thoracic organs from donors harboring occult malignancies.

Post-transplant lymphoproliferative disease in children.

Epstein-Barr virus (EBV)-driven post-transplant lymphoproliferative disease (PTLD) is an important cause of morbidity and mortality following transplantation, and it occurs more frequently in children than in adults. Of 22 (5%) children at our institution who developed tissue-proven PTLD 1-60 months (mean 16.5 months) following organ transplant, 11 died: nine of these 22 patients developed PTLD between 1989 and 1993, and seven (78%) died; the remaining 13 developed PTLD between 1994 and 1998, and four (31%) died (p = 0.08). All nine patients who developed PTLD < 6 months after transplant died, but 11 of 13 patients who manifested disease > or = 6 months after transplant survived (p = 0.0002). Ten of 11 (91%) survivors, but only two of eight (25%) children who died, had serologic evidence of EBV infection at the time of PTLD diagnosis (p = 0.04). EBV seroconversion identified patients at risk for developing PTLD, but also characterized patients with sufficient immune function to survive EBV-related lymphoid proliferation. In situ hybridization for EBER1 mRNA was diagnostically helpful because it detected EBV in tissue sections of all 20 patients with B-cell PTLD, including those with negative serology.

Autopsy pathology of pediatric posttransplant lymphoproliferative disorder.

OBJECTIVES: Posttransplant lymphoproliferative disorder (PTLD) causes significant morbidity and mortality, is related to Epstein-Barr virus (EBV) infection, and is more common in children than in adults. We reviewed autopsies of children who died with PTLD to compare postmortem with antemortem PTLD histology, to assess the extent of PTLD, to document associated pathology, and to identify cause of death. METHODS: Postmortem examinations were performed on 7 patients after bone marrow (n = 3) or liver (n = 4) transplant. PTLD was classified histologically as hyperplasia or lymphoma. In situ hybridization for EBER1 messenger RNA was performed on tissue samples from all cases. EBV serologies were used to categorize infections as negative, primary, or reactive. RESULTS: PTLD was diagnosed in 5 children 12 to 35 (mean: 22) days before death, and 1.5 to 4 (mean: 3) months after transplant; PTLD was diagnosed in 2 cases at autopsy 2.5 and 4 months after transplant. Postmortem PTLD histology resembled antemortem histology; 5 PTLDs were lymphoma, 1 was hyperplasia, and 1 contained both lymphoma and hyperplasia. EBER1 messenger RNA was detected in 6 B-cell PTLDs, including lesions from patients who did not have EBV serology that indicated active infection. Complete autopsy of 4 patients who died with biopsy-proven PTLD revealed widely disseminated disease, and lymph node, brain, gastrointestinal tract, and kidney were involved in all 4 patients. Cases diagnosed at autopsy were 1 widely disseminated PTLD that had been suspected but not proven antemortem, and 1 PTLD confined to abdominal lymph nodes that was not suspected antemortem. Severe organ dysfunction (renal failure, gastrointestinal hemorrhage) was caused by massive PTLD infiltration in 2 patients. The conditions other than PTLD that contributed to morbidity and death were organ infection (5 cases), infarcts (4 cases), and diffuse alveolar damage (3 cases). CONCLUSIONS: PTLD may occur within weeks after transplant in children. The distribution of PTLD comprises a spectrum from localized and subclinical to widely disseminated and symptomatic. PTLD may cause demise quickly after the onset of signs and symptoms, through massive organ infiltration or associated conditions, such as diffuse alveolar damage. EBV serology may not accurately reflect the presence or extent of PTLD. Autopsy studies of transplant patients are necessary to identify the true incidence, natural history, and response to treatment of PTLD.

SHP-1 expression by malignant small B-cell lymphomas reflects the maturation stage of their normal B-cell counterparts.

SHP-1 is a protein phosphotyrosine phosphatase that plays an important role in modulating intracellular signaling, which regulates cell activation, proliferation, differentiation, and migration. It is a negative regulator of signal transduction induced by a number of cell receptors. Our immunohistochemical examination of paraffin-embedded reactive lymph nodes and lymphoid tissues revealed that B lymphocytes in follicle germinal centers do not express SHP-1. A weak staining of the B cells in the germinal center light zones was detected when an ultrasensitive amplification system was used. In contrast, normal B cells in mantle and marginal zones as well as interfollicular B lymphocytes and plasma cells displayed strong immunoreactivity. This pattern of SHP-1 expression was repeated in small B-cell lymphomas. All cases of mantle cell lymphoma (12 of 12), marginal zone lymphoma (10 of 10), and chronic lymphocytic leukemia/small lymphocytic lymphoma (13 of 13) expressed SHP-1 protein. However, only 1 of 30 cases of grade 1 follicle center cell lymphoma expressed SHP-1. Our observations highlight the biologic functions of SHP-1 and demonstrate that the SHP-1 expression pattern by small B-cell lymphomas reflects the maturation stage of their normal cell counterparts. These results indicate that determination of SHP-1 expression may help in the differential diagnosis of small B-cell lymphomas.

Biopsy features are associated with primary symptoms in interstitial cystitis: results from the interstitial cystitis database study.

The purpose of this study was to investigate associations between bladder biopsy features and urinary symptoms for patients enrolled in the Interstitial Cystitis Database (ICDB) Study. Bladder biopsies were obtained during baseline screening in the ICDB Study and were evaluated for histopathologic features. Multivariable models for nighttime voiding frequency, urinary urgency, and pain were developed, incorporating biopsy features from the most diseased area of the bladder as predictors, adjusting for significant clinical factors, and clinical center variation. Among 204 interstitial cystitis (IC) patients providing biopsy specimens, cystoscopic pathology findings were not statistically associated (P >0.1) with primary IC symptoms, although the presence of Hunner's ulcer (n = 12) was suggestive of increased urinary frequency. Within a multivariable predictive model for nighttime voiding frequency, adjusting for age and minimum volume per void, 4 pathology features were noted: (1) mast cell count in lamina propria on tryptase stain; (2) complete loss of urothelium; (3) granulation tissue in lamina propria; and (4) vascular density in lamina propria on factor VIII (F8) stain were statistically significant (P <0.01). Similarly, in a multivariable model for urinary urgency, minimum volume, and percentage of submucosal granulation tissue remained statistically significant (P <0.01). Finally, the percentage of mucosa denuded of urothelium and the percentage of submucosal hemorrhage remained highly associated (P <0.01) with pain in a multivariable predictive model. The fact that the presence or severity of glomerulations was not selected for any of these predictive models suggests that cystoscopic findings of glomerulations are not predictive of IC symptoms. Furthermore, these results suggest an important role for certain pathologic features in the predictive modeling of IC symptoms.

Reduction in immunosuppression as initial therapy for posttransplant lymphoproliferative disorder: analysis of prognostic variables and long-term follow-up of 42 adult patients.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is an Epstein-Barr virus-associated malignancy that occurs in the setting of pharmacologic immunosuppression after organ transplantation. With the increased use of organ transplantation and intensive immunosuppression, this disease is becoming more common. We explore reduction in immunosuppression as an initial therapy for PTLD. METHODS: We analyzed our organ transplant patient database to identify patients with biopsy-proven PTLD who were initially treated with reduction of their immunosuppressive medications with or without surgical resection of all known disease. RESULTS: Forty-two adult patients were included in this study. Thirty patients were treated with reduction in immunosuppression alone. Twelve patients were treated with both reduction in immunosuppression and surgical resection of all known disease. Thirty-one of 42 patients (73.8%) achieved a complete remission. Of those patients who were treated with reduction in immunosuppression alone, 19 of 30 (63%) responded with a median time to documentation of response of 3.6 weeks. Multivariable analysis showed that elevated lactate dehydrogenase (LDH) ratio, organ dysfunction, and multi-organ involvement by PTLD were independent prognostic factors for lack of response to reduction in immunosuppression. In patients with none of these poor prognostic factors, 16 of 18 (89%) responded to reduction in immunosuppression in contrast to three of five (60%) with one risk factor and zero of seven (0%) with two to three factors present. The analysis also showed that increased age, elevated LDH ratio, severe organ dysfunction, presence of B symptoms (fever, night sweats, and weight loss), and multi-organ involvement by PTLD at the time of diagnosis are independent prognostic indicators for poor survival. With median follow-up of 147 weeks, 55% of patients are alive with 50% in complete remission. CONCLUSIONS: Reduction in immunosuppression is an effective initial therapy for PTLD. Clinical prognostic factors may allow clinicians to identify which patients are likely to respond to reduction in immunosuppression.

Isolation and characterization of the TERE1 gene, a gene down-regulated in transitional cell carcinoma of the bladder.

We have identified a novel cDNA product designated transitional epithelial response gene (TERE1), which was localized to chromosome 1p36. The TERE1 transcript (1.5 and 3.5 kb) is present in most normal human tissues including urothelium, but was reduced or absent in the majority of muscle invasive TCC tumors (22 out of 29 cases). The open reading frame encodes a protein of 338 amino acids (MW 36.8 KD). This protein is 57% homologous to a Drosophila protein called heix. We have shown by Western blotting and immuno-histochemistry with a polyclonal antibody to a specific TERE1 peptide, reduced or absent staining in muscle invasive tumors. Transfection of a sense TERE1 construct resulted in an 80-90% inhibition of cellular proliferation in two TCC cell lines and a lack of aneuploidy in the TERE1-transduced J82 cell line. These data suggest a potential role for this gene product in the progression of bladder cancer.

In vitro human tissue models in risk assessment: report of a consensus-building workshop.

Advances in the technology of human cell and tissue culture and the increasing availability of human tissue for laboratory studies have led to the increased use of in vitro human tissue models in toxicology and pharmacodynamics studies and in quantitative modeling of metabolism, pharmacokinetic behavior, and transport. In recognition of the potential importance of such models in toxicological risk assessment, the Society of Toxicology sponsored a workshop to evaluate the current status of human cell and tissue models and to develop consensus recommendations on the use of such models to improve the scientific basis of risk assessment. This report summarizes the evaluation by invited experts and workshop attendees of the current status of such models for prediction of human metabolism and identification of drug-drug interactions, prediction of human toxicities, and quantitative modeling of pharmacokinetic and pharmaco-toxicodynamic behavior. Consensus recommendations for the application and improvement of current models are presented.