Noninvasive papillary urothelial neoplasia (NIPUN): Renaming cancer.

Papillary urothelial neoplasm of low malignant potential (PUNLMP) terminology remains controversial given its reported recurrence rate, its low interobserver diagnostic reproducibility, and its morphologic and molecular genetic overlap with low-grade noninvasive papillary urothelial carcinoma. By contrast, referring to any noninvasive tumor as a "carcinoma" is also controversial. PUNLMP and low-grade noninvasive papillary urothelial carcinomas cannot be reliably distinguished from one another even by experienced pathologists. As both tumors are treated in an identical manner and have similar rates of recurrence and progression, attempting to make this distinction is unnecessary and of little clinical value. These tumor types should therefore be combined into a single category for grading purposes. We propose that all tumors currently classified as either PUNLMP or low-grade noninvasive papillary urothelial carcinoma be termed low-grade noninvasive papillary urothelial neoplasms (NIPUN). This could improve interobserver reproducibility without sacrificing the prognostic utility of histologic grading. PUNLMP terminology should be discontinued and the term "carcinoma" should be reserved only for tumors showing histologic evidence of invasion.

Reappraisal of the papillary urothelial neoplasm of low malignant potential (PUNLMP).

Although the papillary urothelial neoplasm of low malignant potential (PUNLMP) diagnostic category was retained in the updated 2016 World Health Organisation (WHO) classification of tumours of the urinary system, there still exists a great deal of controversy regarding the biological behaviour of these tumours. We review PUNLMP tumours and histological grading with an emphasis on the histomorphological, genetic and clinical similarities between PUNLMP and low-grade non-invasive papillary urothelial carcinoma. A literature search using PubMed was performed. All relevant literature concerning PUNLMP and the grading of urothelial tumours was reviewed. PUNLMPs cannot be reliably distinguished from low-grade non-invasive papillary urothelial carcinomas based on the histomorphological criteria outlined in the WHO 2004/2016 classification system. PUNLMPs and low-grade non-invasive papillary urothelial carcinomas are not only morphologically similar, but also share similar molecular genetic alterations and a similar risk of recurrence and progression. In addition, there are no consensus recommendations for a different method of treatment and follow-up for these two tumour types. Attempting to distinguish PUNLMP from low-grade papillary urothelial carcinoma adds an unnecessary level of complexity to the grading and classification of urothelial tumours. We feel that PUNLMP terminology should be abandoned and that all such tumours should be classified as low-grade carcinomas until more objective determinants of clinical outcome can be established.

Sampling frequency for water quality variables in streams: Systems analysis to quantify minimum monitoring rates.

Insufficient temporal monitoring of water quality in streams or engineered drains alters the apparent shape of storm chemographs, resulting in shifted model parameterisations and changed interpretations of solute sources that have produced episodes of poor water quality. This so-called 'aliasing' phenomenon is poorly recognised in water research. Using advances in in-situ sensor technology it is now possible to monitor sufficiently frequently to avoid the onset of aliasing. A systems modelling procedure is presented allowing objective identification of sampling rates needed to avoid aliasing within strongly rainfall-driven chemical dynamics. In this study aliasing of storm chemograph shapes was quantified by changes in the time constant parameter (TC) of transfer functions. As a proportion of the original TC, the onset of aliasing varied between watersheds, ranging from 3.9-7.7 to 54-79 %TC (or 110-160 to 300-600 min). However, a minimum monitoring rate could be identified for all datasets if the modelling results were presented in the form of a new statistic, ΔTC. For the eight H+, DOC and NO3-N datasets examined from a range of watershed settings, an empirically-derived threshold of 1.3(ΔTC) could be used to quantify minimum monitoring rates within sampling protocols to avoid artefacts in subsequent data analysis.

Characterisation of a Novel Anti-CD52 Antibody with Improved Efficacy and Reduced Immunogenicity.

Anti-CD52 therapy has been shown to be effective in the treatment of a number of B cell malignancies, hematopoietic disorders and autoimmune diseases (including rheumatoid arthritis and multiple sclerosis); however the current standard of treatment, the humanized monoclonal antibody alemtuzumab, is associated with the development of anti-drug antibodies in a high proportion of patients. In order to address this problem, we have identified a novel murine anti-CD52 antibody which has been humanized using a process that avoids the inclusion within the variable domains of non-human germline MHC class II binding peptides and known CD4+ T cell epitopes, thus reducing its potential for immunogenicity in the clinic. The resultant humanized antibody, ANT1034, was shown to have superior binding to CD52 expressing cells than alemtuzumab and was more effective at directing both antibody dependent and complement dependent cell cytotoxicity. Furthermore, when in the presence of a cross-linking antibody, ANT1034 was more effective at directly inducing apoptosis than alemtuzumab. ANT1034 also showed superior activity in a SCID mouse/human CD52 tumour xenograft model where a single 1 mg/Kg dose of ANT1034 led to increased mouse survival compared to a 10 mg/Kg dose of alemtuzumab. Finally, ANT1034 was compared to alemtuzumab in in vitro T cell assays in order to evaluate its potential to stimulate proliferation of T cells in peripheral blood mononuclear cells derived from a panel of human donors: whereas alemtuzumab stimulated proliferation in a high proportion of the donor cohort, ANT1034 did not stimulate proliferation in any of the donors. Therefore we have developed a candidate therapeutic humanized antibody, ANT1034, that may have the potential to be more efficacious and less immunogenic than the current standard anti-CD52 therapy.

First dynamic model of dissolved organic carbon derived directly from high-frequency observations through contiguous storms.

The first dynamic model of dissolved organic carbon (DOC) export in streams derived directly from high frequency (subhourly) observations sampled at a regular interval through contiguous storms is presented. The optimal model, identified using the recently developed RIVC algorithm, captured the rapid dynamics of DOC load from 15 min monitored rainfall with high simulation efficiencies and constrained uncertainty with a second-order (two-pathway) structure. Most of the DOC export in the four headwater basins studied was associated with the faster hydrometric pathway (also modeled in parallel), and was soon exhausted in the slower pathway. A delay in the DOC mobilization became apparent as the ambient temperatures increased. These features of the component pathways were quantified in the dynamic response characteristics (DRCs) identified by RIVC. The model and associated DRCs are intended as a foundation for a better understanding of storm-related DOC dynamics and predictability, given the increasing availability of subhourly DOC concentration data.

Laser-assisted microdissection in translational research: theory, technical considerations, and future applications.

Molecular profiling already exerts a profound influence on biomedical research and disease management. Microdissection technologies contribute to the molecular profiling of diseases, enabling investigators to probe genetic characteristics and dissect functional physiology within specific cell populations. Laser-capture microdissection (LCM), in particular, permits collation of genetic, epigenetic, and gene expression differences between normal, premalignant, and malignant cell populations. Its selectivity for specific cell populations promises to greatly improve the diagnosis and management of many human diseases. LCM has been extensively used in cancer research, contributing to the understanding of tumor biology by mutation detection, clonality analysis, epigenetic alteration assessment, gene expression profiling, proteomics, and metabolomics. In this review, we focus on LCM applications for DNA, RNA, and protein analysis in specific cell types and on commercially available LCM platforms. These analyses could clinically be used as aids to cancer diagnosis, clinical management, genomic profile studies, and targeted therapy. In this review, we also discuss the technical details of tissue preparation, analytical yields, tissue selection, and selected applications using LCM.

KRAS mutation is present in a small subset of primary urinary bladder adenocarcinomas.

AIMS: To determine whether KRAS mutations occur in primary bladder adenocarcinoma. METHODS AND RESULTS: Twenty-six cases of primary urinary bladder adenocarcinoma were analysed. DNA was extracted from formalin-fixed, paraffin-embedded tissue and amplified with shifted termination assay technology, which recognizes wild-type or mutant target sequences and selectively extends detection primers with labelled nucleotides. A mutation in KRAS was found in three (11.5%) of 26 primary bladder adenocarcinomas. Two of these three cases exhibited a G13D mutation, whereas the remaining case contained a mutation in G12V. None of the ten cases of urothelial carcinoma with glandular differentiation displayed KRAS mutation. Colonic adenocarcinoma contained a KRAS mutation in 18 (33%) of 55 cases. There was no distinct difference with regard to grade, stage or outcome according to the limited clinicopathological data available. However, the two youngest patients, aged 32 and 39 years, in our study group, with a mean population age of 61 years, were found to have mutations in KRAS. CONCLUSIONS: KRAS mutations are present in a small subset of primary urinary bladder adenocarcinomas. Future clinical trials for treatment of bladder adenocarcinoma, employing targeted therapies similar to those used for treatment of colon cancer, may also benefit from the predictive implications of KRAS mutational testing.

Discovery of substituted benzyl tetrazoles as histamine H3 receptor antagonists.

A series of potent and subtype selective H3 receptor antagonists containing a novel tetrazole core and diamine motif is reported. A one-pot multi-component Ugi reaction was utilised to rapidly develop the structure-activity relationships (SAR) of these compounds. Optimisation for liver microsome stability (t(1/2)>60 min), minimal CYP inhibition (IC(50)>50 microM) and high cell permeability (Caco-2 P(app) >20x10(-6) cm/s) identified several compounds with drug-like properties.

Does the size matter?: Prostate weight does not predict PSA recurrence after radical prostatectomy.

Previous studies suggest that low prostate weight is a significant negative prognostic factor for prostate cancer. In the current study, the data for 431 men who underwent radical retropubic prostatectomy between 1990 and 1998 were analyzed for association between prostate weight and various clinical and pathologic parameters. These included age, preoperative prostate-specific antigen (PSA) level, PSA recurrence, pathologic stage, Gleason grade, extraprostatic extension, positive surgical margins, tumor volume, associated high-grade prostatic intraepithelial neoplasia, perineural invasion, and lymph node metastasis. Potential associations were probed by using Cox regression model analysis. A significant positive correlation was found between prostate weight and increasing patient age or increasing preoperative PSA level. There was no significant independent association between prostate weight and any of the other variables examined. No association was found between prostate weight and PSA recurrence. Although increasing prostate weight correlates with increased patient age and higher preoperative PSA level, it does not independently predict postoperative cancer recurrence.

Evidence for common clonal origin of multifocal lung cancers.

BACKGROUND: Lung cancer is the most common cause of cancer death in the United States. Multiple anatomically separate but histologically similar lung tumors are often found in the same patient. The clonal origin of multiple lung tumors is uncertain. METHODS: We analyzed 70 lung tumors from 30 patients (23 females and seven males) who underwent surgical resection for lung epithelial tumors, of whom 26 had non-small cell carcinomas and four had carcinoid/atypical carcinoid tumors. All patients had multiple tumors (two to five) involving one or both lungs. Genomic DNA was extracted from paraffin-embedded tissue sections using laser capture microdissection and analyzed for loss of heterozygosity, TP53 mutations, and X-chromosome inactivation status. The percentage (95% confidence interval [CI]) of patients in whom there were concordant patterns of genetic alteration was calculated. RESULTS: All 30 case subjects showed loss of heterozygosity (LOH) in at least one and at most four of the six polymorphic microsatellite markers. Completely concordant LOH patterns between synchronous and metachronous cancers in individual patients were seen in 26 (87%) of 30 informative patients (95% CI = 75% to 99%). Identical point mutations were present in eight of 10 patients who exhibited TP53 mutation by sequencing. Tumors in 18 (78%) of 23 female patients (95% CI = 67% to 98%) showed identical X-chromosome inactivation patterns. Combining the results of LOH studies, TP53 mutation screening analyses, and X-chromosome inactivation data, we demonstrated that the multiple separate tumors in 23 (77%) of 30 patients (95% CI = 62% to 92%) had identical genetic changes, consistent with monoclonal origin of the separate tumors. CONCLUSIONS: Our data indicate that the great majority of multifocal lung cancers have a common clonal origin and that multifocality in lung cancer represents local and regional intrapulmonary metastasis.

New approaches to prediction of immune responses to therapeutic proteins during preclinical development.

Clinical studies show that immunogenicity observed against therapeutic proteins can limit efficacy and reduce the safety of the treatment. It is therefore beneficial to be able to predict the immunogenicity of therapeutic proteins before they enter the clinic. Studies using deimmunized proteins have highlighted the importance of T-cell epitopes in the generation of undesirable immunogenicity. In silico, in vitro, ex vivo and in vivo methods have therefore been developed that focus on identification of CD4+ T-cell epitopes in the sequence of therapeutic proteins. A case study of existing therapeutic proteins is presented to review these different approaches in order to assess their utility in predicting immunogenic potential.

Urothelial carcinoma with an inverted growth pattern can be distinguished from inverted papilloma by fluorescence in situ hybridization, immunohistochemistry, and morphologic analysis.

Inverted papilloma of the urinary bladder and urothelial carcinoma with an inverted (endophytic) growth pattern may be difficult to distinguish histologically, especially in small biopsies. The distinction is important as these lesions have very different biologic behaviors and are treated differently. We examined histologic features and undertook immunohistochemical staining and UroVysion fluorescence in situ hybridization (FISH) to determine whether these methods could aid in making this distinction. We examined histologic sections from 15 inverted papillomas and 29 urothelial carcinomas with an inverted growth pattern. Each tumor was stained with antibodies to Ki-67, p53, and cytokeratin 20. In addition, each tumor was examined with UroVysion FISH for gains of chromosomes 3, 7, and 17 and for loss of chromosome 9p21 signals. None of the inverted papillomas stained positively for Ki-67 or for cytokeratin 20. Only 1 of 15 inverted papillomas stained positively for p53. By contrast, 66%, 59%, and 59% of urothelial carcinomas with an inverted growth pattern stained positively for Ki-67, p53, and cytokeratin 20, respectively. Only 3 of the urothelial carcinomas stained negatively for all 3 immunohistochemical markers. UroVysion FISH produced normal results for all cases of inverted papilloma. By contrast, 21 of 29 cases (72%) of urothelial carcinoma with an inverted growth pattern demonstrated chromosomal abnormalities typical of urothelial cancer and were considered positive by UroVysion FISH criteria. Morphologic features, as well as immunohistochemical stains (including stains for Ki-67, p53, and cytokeratin 20) and/or UroVysion FISH can help to distinguish inverted papilloma from urothelial carcinoma with an inverted growth pattern.

Clonal relationships between epidermotropic metastatic melanomas and their primary lesions: a loss of heterozygosity and X-chromosome inactivation-based analysis.

Loss of heterozygosity (LOH) has previously been demonstrated at multiple chromosome microsatellites in primary and metastatic melanomas. Epidermotropic metastases of melanoma are unique in their varied histopathologic appearance, which can mimic a primary lesion. Our objective was to compare LOH profiles in primary and epidermotropic metastatic melanoma to delineate their clonal relationship. We examined the pattern of allelic loss in the primary melanomas of nine patients in addition to the 21 corresponding epidermotropic metastatic melanomas (average 2.3 metastases per patient). DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser capture microdissection. Eight DNA microsatellite markers on six different chromosomes were analyzed: D1S214 (1p), D6S305 (6q), D9S171 (9p), D9S157 (9p), IFNA (9p), D10S212 (10q), D11S258 (11q), D18S70 (18q). In addition, X-chromosome inactivation analysis was performed in tumors from four women. LOH was seen in 67% (6/9) of primary melanomas and 81% (17/21) of epidermotropic metastatic melanomas. The most frequent allelic losses in informative cases occurred at 10q (33%), 9p (22%), and 11q (22%) in primary melanomas, and at 10q (50%), 1p (44%), and 6q (39%) in epidermotropic metastatic melanomas. Primary lesions demonstrating LOH had concordant allelic loss in at least one locus in a corresponding epidermotropic metastatic melanoma in 83% (5/6) of cases. X-chromosome analysis showed nonrandom inactivation in 75% (3/4) and 71% (5/7) of primary melanoma and epidermotropic metastatic melanoma cases, respectively. Our LOH and X-chromosome inactivation analysis data suggest that epidermotropically metastatic melanomas are clonally related to their primary lesion in many cases. Our data also indicated that some cases diagnosed as epidermotropic metastatic melanoma might be divergent clones or new primaries rather than metastatic disease.

Genetically heterogeneous and clonally unrelated metastases may arise in patients with cutaneous melanoma.

Melanoma of the skin frequently metastasizes to multiple regional lymph nodes and to distant sites. It is uncertain whether all metastases originate from the same tumor clone or whether the genetic heterogeneity of the primary tumor is reflected in the multiple metastases. A total of 73 archival, formalin-fixed, paraffin-embedded, melanoma lesions, including 13 primary tumors and 60 metastases, were studied from 13 patients each having 2 or more metastatic tumors. Genomic DNA samples were prepared from tissue sections using laser-assisted microdissection. We find that the majority of melanoma metastases share a common clonal origin with the matched primary tumor. However, significant genetic divergence occurs frequently during the clonal evolution of metastatic melanoma. In addition, using X-chromosome inactivation analysis, we find that, in some cases, multiple coexisting metastases seem to be derived from different, genetically unrelated tumor clones, implying that some primary tumors may arise from more than a single transformed melanocyte.

Identification and removal of immunogenicity in therapeutic proteins.

The development of anti-therapeutic protein immune responses in patients can be a severe complication of treatment with this class of pharmaceuticals. Antibodies generated against therapeutic proteins limit the clinical efficacy of these agents by neutralizing their biological activity and/or enhancing their clearance. An assessment of the propensity of protein therapeutics to elicit immune responses in patients is likely to become an essential part of their preclinical development. It is clear that CD4+ T-cells are an important factor in the development of long-lived, class-switched, high-affinity antitherapeutic protein antibodies. The increased risk of immunogenicity that is attributed to the presence of T-cell epitopes in therapeutic protein sequences has led to the development of a variety of methods for locating T-cell epitopes and identifying binding peptide amino acids that are important for interacting with either major histocompatibility complex class II molecules or the T-cell receptors. Manipulation of these key residues to disrupt these interactions while maintaining biological activity can result in modified therapeutic proteins with reduced immunogenicity.

Amplifications of EGFR gene and protein expression of EGFR, Her-2/neu, c-kit, and androgen receptor in phyllodes tumor of the prostate.

Phyllodes tumor of the prostate is a rare neoplasm with an unpredictable clinical behavior. It may undergo early recurrence with sarcomatous transformation or may even metastasize. Because targeted therapies have shown great success against several malignancies, there is hope that these same therapies may show similar promise in the treatment of other neoplasms. This study was undertaken to investigate both amplification of the epidermal growth factor receptor (EGFR) gene by fluorescence in situ hybridization and the overexpression of EGFR, Her-2/neu, CD117 (c-kit), and androgen receptor by immunohistochemical staining in a series of 11 phyllodes tumors of the prostate. In the stromal elements, EGFR gene amplification was present in four of 11 tumors and polysomy chromosome 7 was present in two of 11 tumors. No amplification was present in the epithelial components. Only one of 11 tumors had polysomy of chromosome 7 in the epithelial components. Immunohistochemically, in the stromal components, EGFR expression was demonstrable in four of 11 tumors and androgen receptor was demonstrated in six of 10 tumors. Neither Her-2/neu nor c-kit expression was seen in the stromal components of any of the 11 tumors. In the epithelial components, EGFR expression was present in all 11 tumors with strong staining in the basal cell layers and weak or no staining in luminal epithelium; androgen receptor expression was seen in seven of 10 tumors; Her-2/neu was weakly positive in four of 11 tumors; and c-kit expression was present focally and weakly in two of 11 cases with only 2-5% of cells staining. The highest staining intensity and the highest percentage of positively staining cells were seen with EGFR immunostaining in both the stromal and epithelial (mainly basal cells) components. Androgen receptor staining showed the next highest staining intensity and percentage of positive cells in both components. Her-2/neu and c-kit were only weakly or infrequently expressed in the epithelial components of prostatic phyllodes tumors. Our data indicate that EGFR and androgen receptor are frequently and strongly expressed in both epithelial and stromal components of prostatic phyllodes tumors. EGFR gene amplification is frequently present in prostatic phyllodes tumors and may account for one of the mechanisms leading to protein overexpression in some but not all cases. Anti-EGFR and/or antiandrogen agents may be potentially useful for management of patients with tumors expressing EGFR and/or androgen receptor.

Screening for intratubular germ cell neoplasia of the testis using OCT4 immunohistochemistry.

Specific populations of patients are at high risk for the development of germ cell neoplasia. OCT4 has been shown to be a sensitive and specific marker for intratubular germ cell neoplasia of the testis. Whether or not OCT4 immunohistochemistry is a clinically useful screening tool in patients at risk for developing malignant germ cell tumors is not currently known. We undertook immunohistochemical staining for OCT4 in a large series of patients who underwent testicular biopsy or orchiectomy for reasons other than for management of a testicular mass suspicious for malignancy (infertility, cryptorchidism, atrophic testicle, etc.). OCT4 nuclear staining was identified in germ cells in 6 of 157 patients, all of whom had clinical risk factors for the development of testicular germ cell tumors. Two of the 6 patients were under 1.5 years of age, making the significance of OCT4 positivity less certain in these cases. The remaining patients with OCT4-positive germ cells consisted of 3 adults and 1 7-year-old child. Intratubular germ cell neoplasia was identified by light microscopy in only 1 of the 6 OCT4-positive cases. OCT4 immunostaining was negative in all patients who presented with infertility and who had no additional germ cell tumor risk factors. OCT4 immunohistochemistry may be useful in identifying early forms of preinvasive germ cell neoplasia in patients with risk factors for the development of malignant testicular germ cell tumors. The low incidence of OCT4 positivity in the adult infertility patients argues against the routine use of OCT4 immunostains in testicular biopsies for infertility unless additional risk factors are present.