Multitarget Fluorescence Research Articles

Multitarget Fluorescence In Situ Hybridization Diagnostic Applications in Tumors.

Multitarget fluorescence in situ hybridization (mFISH) is a technique that allows the detection of multiple target sequences on the same sample using spectrally distinct fluorophore labels. The mFISH approach is currently a useful assay in the oncologic field for the detection of predictive, prognostic, and diagnostic biomarkers. In this chapter, we summarize the application of mFISH in the identification of target genetic aberrations in formalin-fixed, paraffin-embedded (FFPE) tissue samples of several tumor types. We discuss the mFISH protocols in FFPE samples, the innovative multitarget probes used, and the critical issues related to their interpretation.

Smartphone-assisted colorimetric dual-mode sensing system based on europium-doped metal–organic frameworks for rapid on-site visual detection of Fe3+ and doxycycline

Exploring a rapid, sensitive, low-cost, in-situ intelligent monitoring multi-target fluorescence detection platform is important for food safety and environmental monitoring. A dual-mode ratiometric fluorescence sensing system integrated with a smartphone based on a luminescent metal–organic framework (NH2-MIL-53) and CdTe/Eu was developed for visual, in-situ analysis of Fe3+ and doxycycline (DOX) in this paper. Interestingly, with increasing Fe3+ concentration, the fluorescence sensing system exhibits dual-emission with CdTe QDs at 540 nM as the response signal and NH2-MIL-53 at 438 nm as the reference signal, resulting in a significant color shift of fluorescence color from blue-green to blue, with a linear range of 5––1550 nM and a detection limit of 1.08 nM. In the presence of DOX, the blue fluorescence of NH2-MIL-53 and the green fluorescence of CdTe QDs were quenched respectively by the internal filtering effect and the photoelectron transfer effect. While DOX enhances the red fluorescence of Eu3+ by the antenna effect, forming a triple-emission fluorescence sensor. The visual color of this fluorescent sensor shifted from blue green to grey to pink-white to pink to fuchsia to red as the DOX concentration increased with a detection limit of 0.11 nM. Furthermore, the developed intelligent sensing platform achieved real-time in-situ detection of Fe3+ and DOX with detection limit of 1.47 nM and 6.43 nM, respectively. The platform was applied to detection actual samples with satisfactory results, which proved a promising application for real-time on-site food safety monitoring and human health monitoring.

Self-quenched Fluorophore Dimers for DNA-PAINT and STED Microscopy.

Super-resolution techniques like single-molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non-covalent, weak affinity-based transient labelling systems. DNA-based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi-target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore-labelled single-stranded oligonucleotides. Here, we introduce short-distance self-quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2-fold. We characterise the optical and thermodynamic properties of fluorophore-dimer single-stranded DNA, and show super-resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background.

Nitrogen-doped MoS2 QDs as fluorescent probes for sequential dual-target detection and their microfluidic logic gate operations

Fluorescence sensors show great potential as molecular logic gates to sensitively detect different analytes in trace level. For achieving multi-target analysis, it is desirable for developing complex molecular logic gates by using multi-target fluorescence sensing system. Herein, novel nitrogen-doped molybdenum disulfide quantum dots (MoS2 QDs) have been synthesized and designed as the fluorescent probes for dual-target detection and microfluidic logic gate operations. Nitrogen-doped MoS2 QDs exhibit fluorescence “on–off-on” characteristics by sequential addition of Fe3+ ions and ascorbic acid (AA). The controllably modulating dispersion or aggregation state of nitrogen-doped MoS2 QDs is successfully obtained by addition of Fe3+ or/and AA. The static quenching mechanism of between nitrogen-doped MoS2 QDs and Fe3+ ions is illustrated in detail. This dual-target fluorescence sensor exhibited good sensitivity and selectivity to Fe3+ and AA, and the limit of detection values were 11 μM and 960 nM, respectively. Inspired by these results, nitrogen-doped MoS2 QDs-based “IMPLY” logic gate is fabricated for intelligent fluorescent sensing of Fe3+ and AA. More importantly, using blue fluorescence of nitrogen-doped MoS2 QDs, fluorescent “IMPLY” logic gate integrating with microfluidic chip is constructed for visually discriminating Fe3+ or/and AA with the naked eyes, which has the advantages of microliter reagent consumption, automated operation and visible readout. This “visible-invisible-visible” photoluminescence property of nitrogen-doped MoS2 QDs and their microfluidic logic gate operations provide a novel strategy for dual-target sensing.

UroVysion Multi-Target Fluorescence in situ Hybridization Assay for the Detection of Malignant Bile Duct Brushing Specimens: A Comparison with Routine Cytology

Objective: Routine bile duct brushing cytology is an important diagnostic tool in the evaluation of bile duct stricture. The purpose of this study was to evaluate the performance of the UroVysion fluorescence in situ hybridization (FISH) assay for the detection of malignant bile duct brushing specimens. Study Design: Thirty-five bile duct brushing specimens were included in the study. The FISH assay utilized the commercially available UroVysion probes. The indeterminate cytology results were considered as negative for statistical analysis. Results: Twenty-two of 35 patients were diagnosed as having malignancy based on tissue diagnosis or clinical progression of disease by image assessment. The sensitivity of routine cytology and FISH for the detection of malignancy was 14% (3/22) and 55% (12/22), respectively (p = 0.003). The specificity of routine cytology and FISH was 100% (13/13) and 62% (8/13), respectively (p = 0.025). The false-positive rate for routine cytology and FISH was 0% (0/13) and 38% (5/13), respectively. Conclusions: Our study shows that FISH is significantly more sensitive than routine cytology for the detection of malignancy in bile duct brushing specimens. However, in our study, the specificity of FISH was poor compared to the excellent specificity of routine cytology. The compromised specificity of FISH may limit its utility in the detection of malignant bile duct brushing specimens.

Lab-on-MOFs: Color-Coded Multitarget Fluorescence Detection with White-Light Emitting Metal-Organic Frameworks under Single Wavelength Excitation.

Multitarget assay with single detection modality from individual stimulation mode has aroused great attention. Here, we realize multitarget detection with the color change as signal source from single white light-emitting metal-organic frameworks (MOFs) under single wavelength excitation for the first time. The white light-emitting MOFs are prepared by carefully tuning the ratio of Eu3+, Tb3+, and Dy3+ ions to obtain trimetal MOFs; 5-boronoisophthalic acid (5-bop) provides additional selectivity as ligand. Based on antenna effect, 5-bop is excited to produce its triplet-state, which sensitizes the Ln3+ ions for the white emission. Thus, all of the fluorescence emission is achieved under single wavelength excitation at 275 nm, while any factors affecting the procedure result in the modulation of emission for diverse wavelengths. The trimetal MOFs display distinctive emission colors after interacting with different targets, including metal ions, anions, small molecule, and even biomolecule as a Lab-on-MOFs system. This system shows higher integration degree and simpler preparation and sensing procedures than other multitarget detection strategies.

Analysis of Chromosomal Alterations in Urothelial Carcinoma.

Here, we describe the use of complementary techniques applicable to different types of samples to analyze chromosomal alterations in urothelial carcinoma. By a conventional chromosome analysis on fresh biopsies, it is possible to delineate the status of ploidy and rough chromosomal aberrations. The multi-target fluorescence in situ hybridization (FISH) UroVysion test, for the rapid detection of chromosomal aneusomy of chromosomes 3, 7, and 17 and/or deletion of 9p21 locus, is applicable to urine specimens as well as to formalin-fixed paraffin-embedded (FFPE) specimens and fresh biopsies. Finally, array comparative genomic hybridization (array-CGH) gives the possibility of analyzing the DNA in a single experiment from a biopsy of the tumor but also from FFPE specimens; this technique is able to detect alterations at the genome level not excluding any chromosome.

Fluorescence in situ hybridization as prognostic predictor of tumor recurrence during treatment with Bacillus Calmette\u2013Gu\xe9rin therapy for intermediate- and high-risk non-muscle-invasive bladder cancer

A significant number of patients with intermediate- or high-risk bladder cancer treated with intravesical Bacillus Calmette–Guérin (BCG) immunotherapy are non-responders to this treatment. Since we cannot predict in which patients BCG therapy will fail, markers for responders are needed. UroVysion® is a multitarget fluorescence in situ hybridization (FISH) test for bladder cancer detection. The aim of this study was to evaluate whether FISH can be used to early identify recurrence during treatment with BCG. In a multicenter, prospective study, three bladder washouts at different time points during treatment (t0 = week 0, pre-BCG, t1 = 6 weeks following TURB, t2 = 3 months following TURB) were collected for FISH from patients with bladder cancer treated with BCG between 2008 and 2013. Data on bladder cancer recurrence and duration of BCG maintenance therapy were recorded. Thirty-six (31.6%) out of 114 patients developed a recurrence after a median of 6 months (range 2–32). No significant association was found between a positive FISH test at t0 or t1 and risk of recurrence (p = 0.79 and p = 0.29). A positive t2 FISH test was associated with a higher risk of recurrence (p = 0.001). Patients with a positive FISH test 3 months following TURB had a 4.0–4.6 times greater risk of developing a recurrence compared to patients with a negative FISH. Patients with a positive FISH test 3 months following TURB and induction BCG therapy have a higher risk of developing tumor recurrence. FISH can therefore be a useful additional tool for physicians when determining a treatment strategy.

Abstract 756: Molecular characterization of triploid and tetraploid urine FISH samples

Abstract Urothelial carcinomas (UC) are characterized, among other aberrations, by chromosome 3, 7, 17 gains and 9p21 (p16) loss. Since UC cells readily exfoliate into the urine, molecular cytogenetics techniques have been used to detect cells consistent with UC diagnosis and follow-up: a multitarget Fluorescence In Situ Hybridization (FISH) test, composed of 3, 7 and 17 centromeres and 9p21 locus multicolor probes, has been proved useful for atypical cells identification in both void urine and lower and upper urinary tract samples before morphological changes are apparent through cytological and histological examination. To date, there are no uniform criteria for urine FISH scoring though the test is commonly considered positive following the UroVysion Bladder Cancer Kit criteria (Abbott Molecular): 4 or more cells with a gain of 2 or more chromosomes (polysomy), and/or 12 or more cells with 9p21 homozygous deletion. This definition of polysomy includes trisomy and tetrasomy classes, but, as tetraploidy may represent cells at S/G2 phase, this criterion may lead to false-positives; moreover it has been described that also inflammation induces tetraploidy, and that small percentages of normal urine cells show trisomy/tetrasomy. On the other hand, triploidy and tetraploidy may have a direct role in tumorigenesis, as an intermediate state in the development of cancer aneuploidy. At the moment little is reported about the clinical correlations of urine FISH cases showing only trisomy/tetrasomy and more data are needed to clarify the diagnostic value of triploid and tetraploid urothelial cells. Aiming to better classify patients with a trisomy/tetrasomy urine FISH result, we extracted genomic DNA from 50 fixed urine cell suspensions, previously tested by urine FISH, and investigated them for the presence of fibroblast growth factor receptor 3 (FGFR3) and telomerase reverse transcriptase (TERT) promoter mutations, which constitute the most recurrent somatic alterations in BC. In particular frequent mutations in exons 7, 10 and 15 of FGFR3 characterize non-muscle invasive UC while mutations in the core promoter region of TERT gene, mainly at -124 and -146 positions from the ATG start site, have emerged as the most frequent somatic genetic alteration in both non-muscle invasive and muscle invasive UC. All specimens were subjected to PCR amplification and Sanger sequencing for FGFR3 exon 7 and 10, and for the C228T and C250T TERT promoter mutations. A preliminary analysis of 20 urine FISH cases showed 4 samples with trisomy/tetrasomy, and 1 of these 4 had a mutation of the TERT promotor. Complete results of the FGFR3 and TERT promoter molecular analysis will be presented and correlated to both FISH results and patients clinical data, in order to evaluate if the presence of FGFR3 and/or TERT promoter mutations in urine samples with FISH detected trisomy/tetrasomy may help to identify patients with a higher risk of recurrence and progression. Citation Format: Lorenza Pecciarini, Valeria De Pascali, Ilaria Francaviglia, Anna Talarico, Chiara Iacona, Massimo Freschi, Maria Giulia Cangi, Claudio Doglioni. Molecular characterization of triploid and tetraploid urine FISH samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 756. doi:10.1158/1538-7445.AM2017-756

Target-to-background separation for spectral unmixing in in-vivo fluorescence imaging

We present a novel fluorescence spectral unmixing based on target-to-background separation preprocessing, which effectively separates the multi-target fluorescence from all background autofluorescence (BF) without any hardware-based BF acquisition and tissue specific BF estimation. Specifically, we first enhance the intrinsic accumulation contrast in target-to-background fluorescence using h-dome transformation; then separate multi-target fluorescence areas from the background in sparse multispectral data utilizing kernel maximum autocorrelation factor analysis; we further use fast marching-based image inpainting method to patch up the removed target fluorescence areas and reconstruct the multispectral BF; with the BF matrix being subtracted from the original data, the multi-target fluorophores are easily unmixed from the subtracted data using multivariate curve resolution-alternating least squares method. In two preliminary in-vivo experiments, the proposed method demonstrated excellent performance to unmix multi-target fluorescences while other state-of-art unmixing methods failed to get desired results.

Pleural mesothelioma. Cytology and molecular diagnostics

The definitive diagnosis of malignant mesothelioma (MM) in effusion cytology is often avoided or reluctantly made by cytology alone. The most probable reason for this skepticism is the lack of expertise in cytology among many pathologists and clinicians. When an effusion specimen is composed of cells with unequivocal cytological features of malignancy that have the morphology and immunophenotype of mesothelial cells, the cytological diagnosis of MM is straightforward. However, in the daily routine difficult cases of atypical mesothelial cells are often encountered and additional methods are required to establish an accurate diagnosis. In contrast to reactive mesothelial cells cells of MMs often harbor chromosomal aberrations, most frequently a polysomy in combination with a 9p21 deletion. These chromosomal aberrations can easily be detected by multitarget fluorescence in situ hybridization (FISH); therefore, FISH allows a reliable distinction between reactive mesothelial cells and MM cells. In order to be able to discriminate between MM and adenocarcinoma, an immunocytochemical panel consisting of different mesothelial and epithelial markers is very helpful. In most inconclusive cases of atypical mesothelial cells the combination of morphology, immunocytochemistry and FISH allows a better distinction between reactive mesothelial cells and MM in effusion cytology.

Multitarget fluorescence in-situ hybridization assays in detecting abnormal cells in bronchial washing and biopsied lung cancer cells

Objective Lung carcinomas are associated with molecular abnormalities. We performed a pilot study to determine the feasibility of an interphase fluorescence in-situ hybridization (FISH) assay for the detection of lung carcinoma in bronchial washing specimens and tissue biopsy. Materials and methods Twenty bronchial washing specimens were analyzed. The LAVysion multicolor probe set, which contains locus-specific probes to 5p15, 7p12 (EGFR), and 8q24 (CMYC) and a centromeric probe to chromosome 6, was used; hTERT gene (5p15) red and control region EGR1 gene (5q31) green probes were also used. Results FISH positivity in the samples of this study was 60% (12/20) for LAVysion probes and only 33.3% (6/20) for hTERT probe. Conclusion The current study has indicated that multitarget FISH assays have shown higher sensitivity in detecting abnormal cells in bronchial washing and biopsied cells from patients with lung cancer; in addition, hTERT gene amplification or high copy number could be a marker for very poor prognosis in early-stage non-small-cell lung cancer patients.

Diagnosis of bladder cancer from the voided urine specimens using multi-target fluorescence in situ hybridization

The present study aimed to evaluate the diagnostic value of chromosomal analysis by fluorescence in situ hybridization (FISH) for bladder cancer in light of the histological diagnosis. Several valuable observations using FISH technologies in voided urine cells were also reported. The multi-target FISH-containing probes for the centromeres of chromosomes 3, 7 and 17 and the 9p21 locus were applied to cytospin specimens prepared from voided urine. Urine samples from 53 bladder cancer patients and 30 patients with benign alterations were used for this study. The histological observations of surgical resection specimens showed that the specificity and sensitivity for the technique were 100.0 and 88.0%, respectively. Statistical analyses showed that there was no significant correlation between FISH-positive rate and the tumor stage/grade (P<0.05). However, the proportion of tumor cells with genetic abnormalities positively correlated with the tumor stage (P<0.01). Furthermore, the number of abnormal cells in muscle-invasive pT2 was significantly higher than that in non-muscle-invasive pTa, pT1 (P<0.01). Of 50 patients with bladder cancer, polysomies of chromosomes 3, 7 and 17 were detected in 84.0, 48.0 and 78.0% of cases, respectively, and loss of the 9p21 gene was detected in 80.0% of cases. In addition, the detailed results from different urine specimens showed that FISH assay was required. FISH assay for chromosomes 3, 7 and 17 and 9p21 has a high specificity and sensitivity in the detection of bladder cancer and may reduce the necessity for cytoscopy treatment.

Cost-Effectiveness of Fluorescence In Situ Hybridization in Patients with Atypical Cytology for the Detection of Urothelial Carcinoma

Patients with atypical cytology and equivocal or negative cystoscopy pose a challenge due to uncertainty about the presence of cancer. We determined the cost-effectiveness of using fluorescence in situ hybridization assays to determine the need for biopsy in patients with atypical cytology and equivocal or negative cystoscopy. Data from 2 large prospective studies evaluating the usefulness of fluorescence in situ hybridization in the setting of atypical cytology to detect urothelial carcinoma were combined. The data were used to calculate sensitivity and specificity for the UroVysion fluorescence in situ hybridization assay in various clinical scenarios. Cost data were obtained from our institution and Medicare reimbursement rates. Evaluations with or without bladder biopsy and with or without upper tract evaluation were considered. The study included 263 patients with atypical cytology and equivocal (62) or negative (201) cystoscopy. In patients with equivocal cystoscopy (assuming biopsy was performed in the operating room) biopsy based on fluorescence in situ hybridization results saved $1,740 per patient ($3,267 vs $1,527 per patient) and avoided 42 biopsies compared to biopsy in all patients. If office based biopsies were used then cost savings using fluorescence in situ hybridization results were $95 per patient. Among patients with negative cystoscopy biopsy based on fluorescence in situ hybridization resulted in costs savings of $2,241 per patient, avoiding 167 biopsies, compared to biopsy in all patients. Assuming office based biopsy, the cost savings were $216 per patient. The decision to perform biopsy based on fluorescence in situ hybridization assay in patients with atypical cytology and equivocal or negative cystoscopy was associated with a significant decrease in bladder cancer associated costs.

Primary mesenchymal chondrosarcoma of the kidney with synchronous implant and infiltrating urothelial carcinoma of the ureter

Primary mesenchymal chondrosarcoma of the kidney is rare, and it shows distinct undifferentiated tumor cells and well differentiated cartilagenous components. Also assident infiltrating urothelial carcinoma of the ureter is an extremely rare cancer. We report a case of primary mesenchymal chondrosarcoma occurring in the left kidney with an ipsilateral and distinct distal ureteric implant, and a coexisting infiltrating urothelial carcinoma of the ureter in a 64-year-old man. Histopathological examination and immunohistochemical studuies showed the classic features of mesenchymal chondrosarcoma in kidney, as well as a few infiltrating urothelial in ureter. Multitarget fluorescence in situ hybridization (FISH) suggested that the development of the urothelial carcinoma in the ureter may be triggered or induced by the chondrosarcoma component. The patient died 2 month after left nephro-ureterectomy. This is the first reported case of a primary mesenchymal chondrosarcoma of the kidney with coexisting infiltrating urothelial carcinoma of the ureter.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1522835667751019

Fluorescence In Situ Hybridization Study of Chromosome Abnormalities of Upper Urinary Tract Urothelial Carcinoma in Paraffin-Embedded Tissue

Urothelial carcinomas arising from the upper urinary tract (renal pelvis and ureter) are rare and few molecular genetic studies of these tumors have been conducted to date. We investigated hyperploidy at chromosomes 3, 7, and 17 using a multitarget fluorescence in situ hybridization system to identify genetic alterations in patients with urothelial carcinomas of the upper urinary tract. Chromosomal aberrations are seen most frequently in the high-grade tumors. A highly significant relationship was found between an increase in the percentage of hyperdiploidy and high grade for each chromosome (chromosome 3, P = 6 × 10(-4); chromosome 7, P = 2 × 10(-4); chromosome 17, P = 6 × 10(-5)). To determine whether these associations were independent for each chromosome, the correlation between percentage of hyperdiploidy for each pair of chromosomes was examined. In each case, the correlation was highly significant (R = 0.89-0.91). No statistically significant association was found between percentage of hyperdiploidy and tumor stage for any chromosome.

Reply

We totally agree with the author of the Editorial Comment that patients with suspect of urothelial cell carcinoma due to symptoms such as painless gross hematuria should undergo a complete workup including cystoscopy, upper urinary tract (UUT) imaging, and urine markers. In these cases, urine markers cannot replace invasive diagnostic measures. But how do we proceed in patients with negative cystoscopy results, normal imaging findings, and a positive urine marker? Current guidelines provide no clear recommendations for these patients. Because of the risk of missing a non-papillary bladder tumor (eg, CIS) by cystoscopy and the high specificity of cytology, the presence of positive cytology results should alert urologists. One option for further diagnosis would be to re-perform 5-aminolaevulinic acid (ALA)–guided cystoscopy, which has been shown to be more sensitive than white-light cystoscopy, particularly for the detection of CIS. 1 Stenzl A. Penkoff H. Dajc-Sommerer E. et al. Detection and clinical outcome of urinary bladder cancer with 5-aminolevulinic acid-induced fluorescence cystoscopy: a multicenter randomized, double-blind, placebo-controlled trial. Cancer. 2001; 117: 938-947 Crossref Scopus (92) Google Scholar , 2 Hungerhuber E. Stepp H. Kriegmair M. et al. Seven years' experience with 5-aminolevulinic acid in detection of transitional cell carcinoma of the bladder. Urology. 2007; 69: 260-264 Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar In case of a negative finding, the performance of UUT urine sampling may help to identify the source of positive cytology. Given our findings, the manipulation associated with this procedure does not have a signficant effect on the result of cytology. In the case of positive UUT cytology results, ureteroscopy would be a further option to rule out UUT urothelial carcinoma. Because of studies showing that fluorescence in situ hybridization (FISH) might have a role as an anticipatory positive marker for urothelial carcinoma both in the primary setting and in the follow-up of patients with known bladder cancer. 3 Yoder B.J. Skacel M. Hedgepeth R. et al. Reflex UroVysion testing of bladder cancer surveillance patients with equivocal or negative urine cytology: a prospective study with focus on the natural history of anticipatory positive findings. Am J Clin Pathol. 2007; 127: 295-301 Crossref PubMed Scopus (158) Google Scholar , 4 Skacel M. Fahmy M. Brainard J.A. et al. Multitarget fluorescence in situ hybridization assay detects transitional cell carcinoma in the majority of patients with bladder cancer and atypical or negative urine cytology. J Urol. 2003; 169: 2101-2105 Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar and because of its relatively high specificity, we also recommend re-evaluation of patients with negative cystocopy and positive FISH results, especially in the case of a second positive urine marker (immunocytology or NMP22) or risk factors for urothelial carcinoma (such as smoking). For uCyt, we do not have a clear recommendation because of the relatively high variability of reported specificities. 5 Budman L.I. Kassouf W. Steinberg J.R. Biomarkers for detection and surveillance of bladder cancer. Can Urol Assoc J. 2008; 2: 212-221 PubMed Google Scholar In patients with isolated increased NMP22 and negative cystoscopy results, the low specificity, especially in the context of mechanical manipulation, urinary tract infection, and hematuria, does not justify further invasive measurements. However, the predictive role of positive urine markers has to be evaluated in larger studies to make clear recommendations, especially in the follow-up of patients with urothelial carcinoma. We also agree that aggressive work-up because of positive urine markers in patients without symptoms or risk factors for urothelial carcinoma should be avoided. Given our experience with these markers, we do not agree that further workup for isolated urine markers, especially in the case of present hematuria and positive FISH, cytology and uCyt, results “never reveals any malignant abnormality.” Editorial CommentUrologyVol. 79Issue 3PreviewThe authors have written a straightforward paper describing the false-positive rates for different urinary markers depending upon whether the urine samples were simply voided or were collected after instrumentation, as well as whether there was any evidence for inflammation at time of collection. Some of the markers were quite resistant and their sensitivities/specificities did not change much, whereas for others—specifically NMP22—their utility drops precipitously. Full-Text PDF

Use of Fluorescence In Situ Hybridization to Predict Response to Bacillus Calmette-Guérin Therapy for Bladder Cancer: Results of a Prospective Trial

No reliable methods currently exist to predict patient response to intravesical immunotherapy with bacillus Calmette-Guérin given after transurethral resection for high risk nonmuscle invasive bladder cancer. We initiated a prospective clinical trial to determine whether fluorescence in situ hybridization results during bacillus Calmette-Guérin immunotherapy can predict therapy failure. Candidates for standard of care bacillus Calmette-Guérin were offered participation in a clinical trial. Fluorescence in situ hybridization was performed before bacillus Calmette-Guérin, and at 6 weeks, 3 months and 6 months during bacillus Calmette-Guérin therapy with maintenance. Cox proportional hazards regression was used to assess the relationship between fluorescence in situ hybridization results and tumor recurrence or progression. The Kaplan-Meier product limit method was used to estimate recurrence-free and progression-free survival. A total of 126 patients participated in the study. At a median followup of 24 months 31% of patients had recurrent tumors and 14% experienced disease progression. Patients who had positive fluorescence in situ hybridization results during bacillus Calmette-Guérin therapy were 3 to 5 times more likely than those who had negative fluorescence in situ hybridization results to experience recurrent tumors and 5 to 13 times more likely to have disease progression (p <0.01). The timing of positive fluorescence in situ hybridization results also affected outcomes. For example, patients with a negative fluorescence in situ hybridization result at baseline, 6 weeks and 3 months demonstrated an 8.3% recurrence rate compared to 48.1% for those with a positive result at all 3 points. Fluorescence in situ hybridization results can identify patients at risk for tumor recurrence and progression during bacillus Calmette-Guérin immunotherapy. This information may be used to counsel patients about alternative treatment strategies.

Urovysion: Considerations on modifying current evaluation scheme, including immunophenotypic targeting and locally set, statistically derived diagnostic criteria

Urovysion multitarget fluorescence in situ hybridization (FISH) assay is a promising tool for detection of bladder cancer, however, there is still no consensus regarding abnormal signal pattern and cut-off level, and the recommended targeting carries limitations similar to urine cytology. Aim of this study was to explore diagnostic benefits of a recently introduced method featuring target specific genotyping, as well as to investigate the feasibility of locally and statistically determined cut-off, compared with conventional evaluation scheme. Histology, cytology, and comparative FISH approaches were performed on 42 patients with high clinical suspicion for urothelial carcinoma (UC). FISH parallels were (1) Urovysion-alone (according to manufacturer's instruction); (2) Targeted-Urovysion (cytokeratin7 immunophenotyping followed by Urovysion), both of which evaluated by both conventional and statistical evaluation scheme. For statistical evaluation cut-offs and sufficient sample size were determined on controls and ratio of positive cells was recorded, whereas conventional evaluation relied on manufacturer's recommendations. The specificity of cytology, Urovysion-alone in general and targeted-Urovysion in general appeared 86%, 86%, and 100%, respectively. In the same comparison, overall sensitivity was 60%, 80%, and 93%, respectively. In superficial cases sensitivity was 48% for cytology, 72% for Urovysion-alone and 91% for targeted-Urovysion, while no prominent differences were seen in muscle invasive cases. The ratio of FISH positive cells was proportionate with both stage and grade, however, targeted genotyping could separate high grade/high stage cases more effectively. In conclusion, CK7 targeting raises diagnostic efficiency of Urovysion, and could be an ideal tool for identifying tumor cells in ambiguous cases or when other tumors are present. Statistical evaluation produces accuracy comparable with results of conventional evaluation, and with laboratories setting cut-offs individually but according harmonized protocol, it could aid method standardization. Furthermore, by providing additional quantitative information about tumor characteristics, is likely to have therapy relevant value in the future.

The plasmacytoid carcinoma of the bladder—rare variant of aggressive urothelial carcinoma

The WHO 2004 classification defines new histological and molecular variants of urothelial carcinoma. However, there are limited data available on the clinicopathological characteristics or prognosis of these variants. We present histopathological, molecular and clinical data of 32 plasmacytoid carcinomas of the bladder (PUC) showing that PUC is a high-grade tumor with molecular features of aggressive urothelial carcinoma, usually diagnosed in advanced pathological stage (64% pT3, 23% pT4) showing metastases in 60% of the patients. Average survival of our cohort of PUC treated with radical cystectomy and adjuvant chemotherapy was lower than what is typically seen for comparable conventional urothelial carcinomas. Eighty-seven percent of the PUCs showed a negative or strongly reduced membranous staining of E-cadherin. β-Catenin staining was negative in 22.5%, and 16.7% of the remaining tumors showed nuclear accumulation. Aberrant CK20 expression (negative or >10% of cells stained) and negative CK7 staining was found in 100% and 22.6%, respectively. Ninety-seven percent revealed positive staining for PAN-CK. CD138 was positive in 78%, whereas MUM-1 expression was negative in all cases. Multitarget fluorescence in situ hybridization showed all PUCs to be highly aneuploid and polysomic. Deletions on chromosome 9p21 seem to play an important role in this variant. FGFR3 and PIK3CA mutation analyses yielded no mutations in any of the PUCs analyzed. TP53 mutation analysis showed mutations in 29%. In summary, PUC is an aggressive variant of bladder cancer with molecular features of advanced bladder cancer and evidence of WNT pathway activation in some of the cases.