Clinically Significant Prostate Cancer Detection Following Transrectal and Transperineal Biopsy: Results of the Prostate Biopsy Efficacy and Complications Randomized Clinical Trial.

PURPOSE: The comparative effectiveness of transrectal and transperineal prostate biopsy in detecting clinically significant prostate cancer is not well understood. We conducted a randomized clinical trial to determine whether transperineal biopsy improves the detection of clinically significant prostate cancer. MATERIALS AND METHODS: Of the 840 men randomized, 93% were White, 44% had a previous biopsy, with a median age of 66 years and median PSA density of 0.14. Of these, 384 underwent transrectal and 398 underwent transperineal prostate biopsy. Prebiopsy prostate MRI was performed in 96% of men. Grade Group ≥ 2 prostate cancer was classified as clinically significant. Odds ratios were calculated using logistic regression to evaluate the effect of biopsy procedures on cancer detection rates. RESULTS: The detection rates of clinically significant prostate cancer were 47.1% and 43.2% (odds ratio 1.17; 95% CI, 0.88-1.55) for transrectal and transperineal biopsy, respectively. Age, PSA density, clinical stage and Prostate Imaging Reporting and Data System score were associated with the diagnosis of clinically significant cancer, whereas history of previous biopsy, anterior tumors, and biopsy procedure (transrectal or transperineal) were not. Clinically significant cancer detection rates in biopsy-naïve men undergoing MRI-targeted transrectal or transperineal biopsy were 59% and 62%, respectively. The overall cancer detection rates following transrectal and transperineal biopsy were 72.1% and 70.4%, respectively. CONCLUSIONS: There was no significant difference noted in the detection of clinically significant prostate cancer following transrectal or transperineal prostate biopsy. Urologists may utilize either biopsy procedure that best suits their patients' needs and practice setting.

Complications Following Transrectal and Transperineal Prostate Biopsy: Results of the ProBE-PC Randomized Clinical Trial.

PURPOSE: Transrectal prostate biopsy has come under scrutiny due to potential for postbiopsy infections and transperineal prostate biopsy is being offered as the safer alternative. However, there is a lack of randomized comparative studies. Our goal was to directly evaluate infectious and noninfectious complications following the 2 biopsy procedures. MATERIALS AND METHODS: We conducted a prospective, pragmatic, randomized clinical study in men undergoing prostate biopsy. The participants underwent either transrectal or transperineal prostate biopsy in the office under local anesthesia. The primary outcome was a 30-day composite infectious complication rate, comprising of 1 or more components including fever, genitourinary infection, antibiotic prescriptions, office or emergency visits, hospitalization, or sepsis. Secondary outcomes included 30-day composite noninfectious complications (urinary or hemorrhagic). RESULTS: Of the 763 randomized participants, 718 underwent either transrectal (351) or transperineal (367) prostate biopsy. A composite infectious complication event occurred in 9 participants (2.6%) in the transrectal and 10 participants (2.7%) in the transperineal group (odds ratio, 1.06; 95% CI, 0.43 to 2.65; P = .99). None of the participants developed sepsis in either group. There were no between-group differences in any of the individual component infectious events. A composite noninfectious complication occurred in 6 (1.7%) and 8 (2.2%) participants in the transrectal and transperineal groups, respectively (odds ratio, 1.28; 95% CI, 0.44 to 3.73; P = .79). No participants required hospitalization or other interventions. CONCLUSIONS: Among men undergoing transperineal or transrectal prostate biopsy, we could not demonstrate any difference in the infectious or noninfectious complications. Both biopsy approaches remain clinically viable and safe.

Implementation and Assessment of No Opioid Prescription Strategy at Discharge After Major Urologic Cancer Surgery.

Importance: Postoperative opioid prescriptions are associated with delayed recovery, perioperative complications, opioid use disorder, and diversion of overprescribed opioids, which places the community at risk of opioid misuse or addiction. Objective: To assess a protocol for eliminating postdischarge opioid prescriptions after major urologic cancer surgery. Design, Setting, and Participants: This cohort study of the no opioid prescriptions at discharge after surgery (NOPIOIDS) protocol was conducted between May 2017 and June 2021 at a tertiary referral center. Patients undergoing open or minimally invasive radical cystectomy, radical or partial nephrectomy, and radical prostatectomy were sorted into the control group (usual opioids), the lead-in group (reduced opioids), and the NOPIOIDS group (no opioid prescriptions). Interventions: The NOPIOIDS group received a preadmission educational handout, postdischarge instructions for using nonopioid analgesics, and no routine opioid prescriptions. The lead-in group received a postdischarge instruction sheet and reduced opioid prescriptions at prescribers' discretion. The control group received opioid prescriptions at prescribers' discretion. Main Outcomes and Measures: Primary outcome measures included rate and dose of opioid prescriptions at discharge and for 30 days postdischarge. Additional outcome measures included patient-reported pain and satisfaction level, unplanned health care utilization, and postoperative complications. Results: Of 647 opioid-naive patients (mean [SD] age, 63.6 [10.0] years; 478 [73.9%] male; 586 [90.6%] White), the rate of opioid prescriptions at discharge for the control, the lead-in, and the NOPIOIDS groups was 80.9% (157 of 194), 57.9% (55 of 95), and 2.2% (8 of 358) (Kruskal-Wallis test of medians: P < .001), and the overall median (IQR) tablets prescribed was 14 (10-20), 4 (0-5.3), and 0 (0-0) per patient in the control, lead-in, and NOPIOIDS groups, respectively (Kruskal-Wallis test of medians: P < .001). In the NOPIOIDS group, median and mean opioid dose was 0 tablets for all procedure types, with the exception of kidney procedures (mean [SD], 0.5 [1.7] tablets). Patient-reported pain surveys were received from 358 patients (72.6%) in the NOPIOIDS group, demonstrating low pain scores (mean [SD], 2.5 [0.86]) and high satisfaction scores (mean [SD], 86.6 [3.8]). There was no increase in postoperative complications in the group with no opioid prescriptions. Conclusions and Relevance: This perioperative protocol, with emphasis on nonopioid alternatives and patient instructions, may be safe and effective in nearly eliminating the need for opioid prescriptions after major abdominopelvic cancer surgery without adversely affecting pain control, complications, or recovery.

Distinct mutational status in GATA3-Positive clear cell adenocarcinoma of the urinary tract: A CASE report.

•Clear cell adenocarcinoma of the lower urinary tract is rare and poses diagnostic challenge.•GATA3, which is frequently expressed in urothelial carcinoma, can be expressed in clear cell adenocarcinoma.•ARID1A, PBRM1, ERBB4, and SMARCA4 mutations were identified in the current CASE.•Molecular studies may aid in the diagnosis, and optimal treatment decision-making process.

Rationale and protocol for randomized study of transrectal and transperineal prostate biopsy efficacy and complications (ProBE-PC study).

BACKGROUND: Rrisk of infection and hospitalization after transrectal prostate biopsy (TRBx) has been increasing worldwide. Several modified antibiotic regimens have met with variable success in preventing such infections. Transperineal prostate biopsy (TPBx) is increasingly recommended as the preferred alternative due to a potentially lower risk of post-biopsy infections. Aim of this review is to define the magnitude of post-biopsy complications and the effectiveness of preventive strategies, including TPBx approach. METHODS: We performed a focused review of literature on infectious complications after TRBx and detailed the use of various preventive measures. We summarized the effectiveness of several preventive measures, including TPBx, and outlined the inconsistencies in reported outcomes. We identified potential barriers to the uptake of TPBx, including the gap in knowledge such as lack of high-quality evidence. RESULTS: Several antibiotic prophylaxis protocols, including targeted and augmented, have been utilized for TRBx without demonstrating a clearly superior regimen. Of the non-antibiotic preventive measure, povidone-iodine rectal prep appears to be most effective strategy. Several single-arm cohort studies have reported very low rates of infections after TPBx and demonstrated the feasibility of an office-based procedure. However, barriers to the adoption of TPBx exist including retrospective data, and conflicting results showing minimal reduction in complications with increased burden of resource utilization. Presently, there are no randomized studies comparing the infectious complications after TRBx and TPBx. We discuss the rationale and protocol for a randomized controlled trial to determine the comparative effectiveness of biopsy techniques. CONCLUSIONS: TPBx approach has the potential to lower the rate of post-biopsy infections and hospitalizations. However, there are several barriers to widespread adoption of this approach including inconsistencies in reported outcomes and lack of Level-1 evidence. Randomized controlled studies are required to directly compare the infectious complications associated with each biopsy procedure.

Expression of Small Noncoding RNAs in Urinary Exosomes Classifies Prostate Cancer into Indolent and Aggressive Disease.

PURPOSE: This is the first report of the development and performance of a platform that interrogates small noncoding RNAs (sncRNA) isolated from urinary exosomes. The Sentinel™ PCa Test classifies patients with prostate cancer from subjects with no evidence of prostate cancer, the miR Sentinel CS Test stratifies patients with prostate cancer between those with low risk prostate cancer (Grade Group 1) from those with intermediate and high risk disease (Grade Group 2-5), and the miR Sentinel HG Test stratifies patients with prostate cancer between those with low and favorable intermediate risk prostate cancer (Grade Group 1 or 2) and those with high risk (Grade Group 3-5) disease. MATERIALS AND METHODS: sncRNAs were extracted from urinary exosomes of 235 participants and interrogated on miR 4.0 microarrays. Using proprietary selection and classification algorithms, informative sncRNAs were selected to customize an interrogation OpenArray™ platform that forms the basis of the tests. The tests were validated using a case-control sample of 1,436 subjects. RESULTS: The performance of the miR Sentinel PCa Test demonstrated a sensitivity of 94% and specificity of 92%. The Sentinel CS Test demonstrated a sensitivity of 93% and specificity of 90% for prediction of the presence of Grade Group 2 or greater cancer, and the Sentinel HG Test demonstrated a sensitivity of 94% and specificity of 96% for the prediction of the presence of Grade Group 3 or greater cancer. CONCLUSIONS: The Sentinel PCa, CS and HG Tests demonstrated high levels of sensitivity and specificity, highlighting the utility of interrogation of urinary exosomal sncRNAs for noninvasively diagnosing and classifying prostate cancer with high precision.

Rectal ulcer and pseudomalignant epithelial changes after prostate seed brachytherapy: A rare complication with a diagnostic pitfall.

BACKGROUND: Implant brachytherapy (IBT) is a well-recognized treatment modality for early stage prostate cancer. Rectal ulcer and rectourethral fistula complicating IBT may cause an alteration of the normal anatomic landmarks. In this context, pseudomalignant radiation-induced changes within prostatic epithelium may be misinterpreted as a primary rectal malignancy. Such challenging and misleading findings have not been described, and may not be recognized as such. MATERIALS AND METHODS: We present the clinical and pathologic aspects of two patients who underwent IBT for low stage prostate cancer that was complicated by deep rectal ulcer. Both patients underwent extensive palliative surgical resection for disease control. RESULTS: The histologic changes in both cases were noteworthy for extensive necrosis and inflammation of the prostate, associated with loss of recto-prostatic anatomical landmarks. Prostatic glands showed striking radiation-induced atypia and pseudomalignant epithelial changes extending to the rectal ulcer bed, with no residual viable tumor. The first patient had undergone a biopsy of the rectal ulcer bed that was misinterpreted as a rectal adenocarcinoma prior to surgery. The similarity between atypical glands of the biopsy and the benign prostatic tissue with radiation-induced atypia in resection specimen confirmed their benign nature. CONCLUSIONS: Deep rectal ulcer complicating IBT may lead to distortion of the normal recto-prostatic anatomical landmarks, resulting in detection of pseudo-malignant prostatic glands at the ulcer base. Such findings may be mistaken for a primary rectal malignancy in limited biopsy material if not familiar to the pathologist.

Conformational Changes in the 5' End of the HIV-1 Genome Dependent on the Debranching Enzyme DBR1 during Early Stages of Infection.

Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it.IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.

NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells.

This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from Sambrook and Russell (2001) in order to downscale the amount of cells needed. To determine the efficiency of fractionation, we recommend using qPCR to compare the subcellular compartments that have been purified with equivalent amount of control whole cell extracts.

Impairment of HIV-1 cDNA synthesis by DBR1 knockdown.

UNLABELLED: Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different factors and occur in different intracellular locations. IMPORTANCE: This study shows that HIV-1 reverse transcription starts in the cytoplasm but is completed in or on the surface of the nucleus. Moreover, we show that nuclear reverse transcription is dependent on the activity of the human RNA lariat debranchng enzyme (DBR1), while cytoplasmic reverse transcription is not. These findings may provide new avenues for inhibiting HIV-1 replication and therefore may lead to new medicines for treating HIV-1-infected individuals.

Micropapillary carcinoma: new variant of prostatic acinar adenocarcinoma.

A micropapillary variant of prostatic acinar adenocarcinoma has not been reported in the literature. Herein, we report a case of a 50-year-old patient who presented with an elevated prostate-specific antigen concentration and was subsequently diagnosed with prostatic acinar adenocarcinoma on biopsy. Radical prostatectomy specimen revealed prostatic carcinoma with Gleason score 4 + 5  =  9/10, with micropapillary component constituting 80% of tumor volume. Immunohistochemical studies of the prostate carcinoma showed a homogeneously positive prostate-specific antigen and α-methylacyl-CoA racemase, high-molecular-weight cytokeratin, and p63 protein cocktail pattern of staining in both micropapillary and conventional components. Pelvic lymph nodes were negative for metastatic disease. In contrast to the aggressive behavior of micropapillary carcinomas of other organs, the disease in our patient has thus far followed a more benign course, with low stage on presentation and a 2-year follow-up free of disease. However, prognostic correlation should be established on large series in order to assign this variant to a grade category within the Gleason scheme.

A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins.

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.

Localized lymphedema (elephantiasis): a case series and review of the literature.

BACKGROUND: Lymphedema typically affects a whole limb. Rarely, lymphedema can present as a circumscribed plaque or an isolated skin tumor. OBJECTIVE: To describe the clinical and pathologic characteristics and etiologic factors of localized lymphedema. METHODS: Case-control study of skin biopsy and excision specimens histologically diagnosed with lymphedema and presenting as a localized skin tumor identified during a 4-year period. RESULTS: We identified 24 cases of localized lymphedema presenting as solitary large polyps (11), solid or papillomatous plaques (7), pendulous swellings (4), or tumors mimicking sarcoma (2). Patients were 18 females and 6 males with a mean age of 41 years (range 16-74). Anogenital involvement was most frequent (75%)--mostly vulva (58%), followed by eyelid (13%), thigh (8%) and breast (4%). Causative factors included injury due to trauma, surgery or childbirth (54%), chronic inflammatory disease (rosacea, Crohn's disease) (8%), and bacterial cellulitis (12%). Eighty-five percent of these patients were either overweight (50%) or obese (35%). Compared with a series of 80 patients with diffuse lymphedema, localized lymphedema patients were significantly younger (41 vs. 62 years old, p = 0.0001), had no history of cancer treatment (0% vs. 18%, p = 0.03), and had an injury to the affected site (54% vs. 6%, p = 0.0001). Histologically, all cases exhibited dermal edema, fibroplasia, dilated lymphatic vessels, uniformly distributed stromal cells and varying degrees of papillated epidermal hyperplasia, inflammatory infiltrates and hyperkeratosis. Tumor size significantly and positively correlated with history of cellulitis, obesity, dense inflammatory infiltrates containing abundant plasma cells, and lymphoid follicles (p < 0.05). A history of cellulitis, morbid obesity, lymphoid follicles and follicular cysts predicted recurrent or progressive swelling despite excision (p < 0.05). CONCLUSIONS: Localized lymphedema should be considered in the etiology of skin tumors when assessing a polyp, plaque, swelling or mass showing dermal edema, fibrosis and dilated lymphatics on biopsy. A combination of lymph stasis promoting factors (trauma, obesity, infection and/or inflammatory disorders) produces localized elephantiasis.

Tissue-marking scheme for a cost-effective extended prostate biopsy protocol.

OBJECTIVES: Extended biopsy schemes are now the standard of care for detection of prostate cancer. Submitting biopsy cores individually raises the cost of pathologic evaluation significantly while important prognostic information is lost when the samples are bundled into fewer containers. We devised a protocol for bundling biopsy cores to reduce the cost while maintaining our ability to identify important biopsy features. MATERIALS AND METHODS: Four hundred fifty-two consecutive men underwent a prostate biopsy using our prospectively designed protocol. The lateral peripheral cores were marked with India ink and combined with cores from the corresponding sextant site into one container (maximum containers = 6). Prognostic information from each core was recorded. Cost analysis was based on the reimbursement rates for variable number of containers. RESULTS: Tissue-labeling protocol did not increase the procedure time or introduce any tissue artifacts. Cancer was detected in 177 (39%) men with mean Gleason score of 7. A single core with cancer was noted in 28%, and cancer in < or =25% of the core was found in 41%. Thirteen of 64 (20%) men undergoing radical prostatectomy had extracapsular extension (ECE) and 10 (15%) had a positive surgical margin. The location of ECE on prostatectomy specimen correlated with a positive biopsy site in 9 (70%) patients. The cost of histopathologic evaluation is based on number of individually labeled specimen containers. By reducing the number of specimen containers from 12 to 6, the potential savings may be in hundreds of million per year. CONCLUSIONS: This simple tissue-labeling protocol facilitates extended prostate biopsies in a cost-effective manner, while maintaining our ability to glean important prognostic information from each core.

PSA-related markers in the detection of prostate cancer and high-grade disease in the contemporary era with extended biopsy.

OBJECTIVE: To determine the relationship of total PSA (tPSA), percent free PSA (%fPSA), and complexed PSA (cPSA) with prostate cancer detection and the diagnosis of poorly-differentiated cancers in the contemporary era. METHODS: We retrospectively reviewed the clinical and pathological records of 292 men who met the following inclusion criteria: (1) tPSA 2.5 to 10 ng/ml; (2) initial biopsy only; (3) extended biopsy scheme (>or=10 peripheral zone cores); (4) no previous prostate surgeries. The ability of PSA-related markers to detect cancer was determined by area under the receiver operating characteristics curve analysis (AUC-ROC). Various clinically relevant % fPSA cutoffs and cPSA ranges were analyzed to determine the association with poorly-differentiated cancers. RESULTS: Cancer was detected in 126 (43%) men, with mean Gleason score of 7. The cancer detection rates for various cutoffs of tPSA, cPSA and % fPSA were very similar. On ROC analysis for cancer diagnosis, the AUCs for tPSA, % fPSA, and cPSA were 0.53, 0.54, and 0.52, respectively. Men with % fPSA <15 were more likely to have poorly-differentiated cancer than those with % fPSA >or=15 (66% vs. 41%, P < 0.005). Similarly, cPSA ranges (2-4, 4.1-6, and >6) were associated with the detection of poorly-differentiated cancers (37%, 57%, and 80% P < 0.003). CONCLUSIONS: With the use of extended prostate sampling in the contemporary screening population, the addition of cPSA and % fPSA does not enhance the diagnostic performance of tPSA. However, the significant association between cPSA and poorly-differentiated cancers suggests that this may be a more useful initial test for prostate cancer screening.

Loss of claudins-1 and -7 and expression of claudins-3 and -4 correlate with prognostic variables in prostatic adenocarcinomas.

Claudins, members of a large family of adherent junction proteins, regulate the integrity and function of tight junctions and influence tumorigenesis. Recent studies have shown that altered levels of the different claudins may be related to invasion and progression of carcinoma cells in several primary neoplasms. However, there is no reported study documenting the pattern of claudin expression in prostatic adenocarcinomas (PACs). Formalin-fixed paraffin-embedded sections from 141 PACs were immunostained by manual and automated methods on the Xmatrx (BioGenex, San Ramon, CA) using antihuman claudin-1, -3, -4, -5, and -7 antibodies (Zymed, San Francisco, CA). Membranous immunoreactivity for each protein was semiquantitatively scored in both the tumor and adjacent benign epithelium in each case. Results were correlated with clinicopathologic variables. Variable membranous positivity was noted in the adjacent benign glands for all 5 proteins in all cases. PACs showed variable membranous positivity ranging from decreased, similar to, and increased in relation to the adjacent benign epithelium for all claudins. Decreased expression of claudin-1 correlated with high tumor grade (P = .001) and biochemical disease recurrence (P = .01), whereas decreased claudin-7 correlated with high tumor grade (P < .0001). In contrast, expression of claudin-3 correlated with advanced stage tumors (P = .03) and recurrence (P = .02), and expression of claudin-4 correlated with advanced stage (P = .02). On multivariate analysis, advanced stage (P = .026) and decreased claudin-1 protein expression (P = .005) independently predicted disease recurrence. Immunohistochemical expression and prognostic significance of claudins are variable in PACs, with decreased expression of claudin-1 emerging as an independent prognostic variable warranting further study.

The creation of a novel fluorescent protein by guided consensus engineering.

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.

Antibody binding loop insertions as diversity elements.

In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.