Multiprobe Fluorescence In Situ Hybridization Research Articles

Multi-probe FISH Analysis of Immunophenotyped Chronic Lymphocytic Leukemia by Imaging Flow Cytometry.

Imaging flow cytometry is an automated method that enables cells and fluorescent signals to be visualized and quantified. Here, we describe a new imaging flow cytometry method whereby fluorescence in situ hybridization (FISH) is integrated with cell phenotyping. The method, called "immuno-flowFISH," provides an exciting new dimension for the analysis of genomic changes in cytological samples (e.g., blood, bone marrow). Cells are analyzed in suspension without any requirement for prior cell isolation or separation. Multiple antibodies and FISH probes, each with a unique fluorophore, can be added and many thousands of cells analyzed. Specific cell populations are identified by their antigenic profile and then analyzed for the presence of chromosomal defects. Immuno-flowFISH was applied to the assessment of chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm where chromosomal abnormalities predict prognosis and treatment requirements. This integrated immunophenotyping and multi-probe FISH strategy could detect both structural and numerical chromosomal changes involving chromosomes 12 and 17 in CLL cells. Given that many thousands of cells were analyzed and the leukemic cells were positively identified by their immunophenotype, this multi-probe method adds precision to the cytogenomic analysis of CLL. © 2021 Wiley Periodicals LLC.

Evaluation of multiprobe fluoresence in-situ hybridization panel in the detection of common chromosomal abnormalities of acute myeloid leukemia

Background Numerous recurrent chromosomal aberrations have been identified in acute myeloid leukemia (AML), and their detection has become essential for accurate diagnosis, classification, and prognosis of the disease. Fluorescence in-situ hybridization (FISH) provides a powerful technique complementary and even alternative to chromosome banding studies for the identification of selected chromosome aberrations. Aim Evaluation of the multiprobe FISH panel in the detection of common cytogenetic abnormalities in AML and to investigate their association with clinical diagnosis, chemotherapy, and prognosis. Patients and methods This study was conducted on 20 newly diagnosed AML patients. All patients were subjected to full history taking, clinical examination, laboratory investigations including complete blood count, bone marrow aspiration, immunophenotyping, and interphase FISH using cytocell multiprobe AML/myelodysplastic syndrome panel designed to detect AML/Eight-twenty-one (ETO), Promyelocytic leukaemia- Retinoic acid receptor alpha (PML-RARA), and core-binding factor beta/Myosin, heavy chain 11, smooth muscle (CBFβ/MYH11) translocations, mixed-lineage leukaemia (MLL) break apart, P53, 5q, 7q, and 20q deletions. Results Interphase FISH analysis showed 5q deletion in 8/20 (40%) cases, positive PML/RARA in 3/20 (15%) cases p53 deletion in 15/20 (75%) cases, positive AML1/ETO in 1/20 (5%) cases, no MLL break apart cases (0%), 7q deletion in 4/20 (20%) cases, positive CBFβ/MYH11 fusion gene in 4/20 (20%) cases, 20q deletion in 9/20 (45%) cases, and trisomy 8 in 7/20 (35%) cases. There was a statistically significant relationship between 5q deletion and prognosis (P=0.028). Conclusion Multiprobe FISH is more cost effective and time effective compared with traditional FISH. It is an efficient technique for the detection of cytogenetic aberrations AML, providing critical information for diagnosis and prognosis, and for monitoring the course of the disease.

Targeted multiprobe fluorescence in situ hybridization analysis for elucidation of inconclusive pancreatobiliary cytology.

Endoscopic fine-needle aspiration (FNA) and brush cytology are standard methods for the diagnosis of pancreatobiliary malignancies. Although the majority of cytological diagnoses are straightforward, there remains a difficult category of inconclusive cytology. This study explored the utility of fluorescence in situ hybridization (FISH) to improve the diagnostic stratification between reactive and malignant cells in cases of inconclusive cytology. The multiprobe FISH assay UroVysion was used for copy number assessment of chromosomes 3, 7, 17, and the 9p21 locus on Papanicolaou-stained specimens with a diagnosis of inconclusive cytology (n = 50), adenocarcinoma (n = 31) and no evidence of malignancy (n = 9). The target cells were photographed and their coordinates saved on an automated stage prior to hybridization. A positive test was defined as increased copy number (> 2) of at least 2 chromosomes (3, 7, or 17) in at least 4 atypical cells, or loss of 9p21 in at least 12 cells. FISH confirmed all 31 cytological diagnoses of pancreatobiliary adenocarcinomas, and was negative in the 9 patients with negative cytology. Among the 50 cases with inconclusive cytology, FISH detected 19 of 31 cases with a final diagnosis of adenocarcinoma, and was negative in all 19 cases with no final evidence of malignancy (sensitivity of 61.3%, specificity of 100%, positive predictive value of 100%, negative predictive value of 61.3%). Loss of 9p21 was found in 43 (86%) of all 50 FISH-positive cases. Multiprobe FISH combined with automated relocation of atypical cells is a powerful technique to clarify inconclusive cytology of the pancreatobiliary tract, allowing for a better distinction between reactive atypia and malignancy.

Evaluation of urovysion and cytology for bladder cancer detection

Urine cytology has been used for screening of bladder cancer but has been limited by its low sensitivity. UroVysion is a multiprobe fluorescence in situ hybridization (FISH) assay that detects common chromosome abnormalities in bladder cancers. For this study, the authors evaluated the effectiveness of multiprobe FISH and urine cytology in detecting urothelial cell carcinoma (UCC) in the same urine sample. In total, 1835 cases with the following criteria were selected: valid results from both the multiprobe FISH assay and urine cytology in the same urine sample, histologic and/or cystoscopic follow-up within 4 months of the original tests, or at least 3 years of clinical follow-up information. The results of FISH and cytology were correlated with clinical outcomes derived from a combination of histologic, cystoscopic, and clinical follow-up information. Of 1835 cases, 1045 cases were from patients undergoing surveillance of recurrent UCC, and 790 were for hematuria. The overall sensitivity, specificity, positive predictive value, and negative predictive value in detecting UCC were 61.9%, 89.7%, 53.9%, and 92.4%, respectively, for FISH and 29.1%, 96.9%, 64.4%, and 87.5%, respectively, for cytology. The performance of both FISH and cytology generally was better in the surveillance population and in samples with high-grade UCC. In 95 of 296 cases with atypical cytology that were proven to have UCC, 61 cases, mostly high-grade UCC, were positive using the multiprobe FISH assay. The UroVysion multiprobe FISH assay was more sensitive than urine cytology in detecting UCC, but it produced more false-positive results. The current data suggest that the use of FISH as a reflex test after an equivocal cytologic diagnosis may play an effective role in detecting UCC.

UroVysion® multiprobe FISH in the triage of equivocal urinary cytology cases

The search for biological and clinical significance of equivocal urinary cytology has emerged as the most promising application of fluorescence in situ hybridization (FISH). Using the multiprobe UroVysion(®) assay, a negative FISH result in the presence of atypical urothelial cells favors the presence of reactive, benign alterations and helps to avoid unnecessary invasive procedures. However, a negative FISH result in case of a negative or equivocal cytology does not exclude low-grade urothelial neoplasia. Equivocal findings are a notorious problem after conservative treatment, particularly after BCG immunotherapy of carcinoma in situ, where even benign reactive changes can appear worrisome. In this situation, a positive FISH result in spite of non-high grade cytology independently indicates persistent or recurrent urothelial carcinoma. However, chromosomal abnormalities are not restricted to malignancy but may also occur in benign cells. Tetraploidy with a balanced duplication of the whole genome, or polyploidy can occur in non-neoplastic conditions of the bladder such as Decoy cells, radiotherapy-induced changes and urolithiasis. Thus, a positive FISH result in a patient with a history of pelvic irradiation does not prove cancer unless there is unequivocal 9p21 deletion. Recent studies show that an aggressive workup of patients with a suspicious cytology+positive UroVysion(®) result and negative cystoscopy is not currently justified. However, multi-target UroVysion(®) FISH remains an excellent tool to improve diagnosis in urinary cytopathology, provided that FISH results are interpreted in the light of the clinical situation, and that one reminds that FISH adds no diagnostic value in case of clearly positive, high-grade cytology.

Fluorescence in situ hybridization assay detects upper urinary tract transitional cell carcinoma in patients with asymptomatic hematuria and negative urine cytology

We evaluated the performance of a multiprobe FISH (fluorescence in situ hybridization) assay for noninvasive detection of upper urinary tract transitional cell carcinoma (UUT-TCC) in patients with asymptomatic hematuria and negative urine cytology. Voided urine samples from 285 patients with asymptomatic hematuria and negative urine cytology were prospectively analyzed by FISH technique. FISH assays were performed to detect chromosomal changes frequently associated with TCC, including aneuploidy of chromosomes 3, 7 and 17, and loss of the 9p21 locus. Eleven (3.9%) had a positive FISH result. Of the 11 patients, nine (81.8%) were found to have a TCC of the upper urinary tract, while no patients with negative FISH findings were found to have UUT-TCC. In this selected cohort, the sensitivity and specificity of FISH for the detection of UUT-TCC was 100% and 99.3%, respectively. Our preliminary data suggest that the clinical utility of FISH assay of chromosomes 3, 7, 9, and 17 as a noninvasive ancillary test for the diagnosis of UUT-TCC in a selected patient population with asymptomatic hematuria and negative urine cytology and by significant high sensitivity and specificity may be a reliable diagnostic approach for early detection of UUT-TCC patients. Further larger prospective and multicenter trials are needed to confirm our results.

Semi‐automated scoring of triple‐probe FISH in human sperm: Methods and further validation

Although the frequency and consequence of sperm chromosomal abnormalities are considerable, few epidemiologic studies in large samples have been conducted to investigate etiologic risk factors. This is, in part, attributable to the labor intensive demands of manual sperm fluorescence in situ hybridization (FISH) scoring. As part of an epidemiologic study investigating environmental risk factors for aneuploidy among men attending a hospital-based fertility clinic, a semi-automated method of slide scoring was further validated and used to estimate sex chromosome sperm disomy frequency in a large number of samples. Multiprobe FISH for chromosomes X, Y, and 18 was used to determine sex chromosome disomy in sperm nuclei. Semi-automated scoring methods were used to quantify X disomy (sperm FISH genotype XX18), Y disomy (YY18), and XY disomy (XY18). The semi-automated results were compared with the results from manual scoring in 10 slides. The semi-automated method was then used to estimate sex chromosome disomy frequency in 60 men. Of 10 slides scored, significant differences between the manual and semi-automated results were seen primarily in one slide that was of poor quality because of over swollen nuclei. Among 60 men analyzed using the semi-automated method, median total sex chromosome disomy frequency was 1.65%, which is higher than seen among normal men but within range with reports from fertility clinic populations. These results further validate that semi-automated methods can be used to score sperm disomy with results comparable to manual methods. This is the largest study to date to provide estimates of sex chromosome disomy among men attending fertility clinics. These methods should be replicated in larger clinic populations to arrive at stable estimates of aneuploidy frequency in men who are members of subfertile couples. © 2011 International Society for Advancement of Cytometry.

Clinical Utility of Fluorescence In Situ Hybridization for Prediction of Residual Tumor After Transurethral Resection of Bladder Urothelial Carcinoma

To evaluate the use of a multiprobe fluorescence in situ hybridization (FISH) assay for predicting the residual tumor load after transurethral resection (TUR) of bladder urothelial carcinoma (UC). Voided urine specimens were collected from 125 consecutive patients with suspected UC who had been admitted for TUR. Of the 125 patients, 89 with UC diagnosed underwent a second procedure (repeated TUR or cystectomy) 4-6 weeks after the initial TUR and were included in the present study. Using the pathologic findings from the second procedure, the patients were divided into those with (n = 38) and those without (n = 51) residual tumor. Urine samples were taken both before and after the initial TUR. The multiprobe FISH assay was performed to detect aneuploidy of chromosomes 3, 7, and 17 and the loss of the 9p21 locus. Before the first TUR, no significant difference was found in the FISH-positive percentage between those with and without residual tumor. After the first TUR, the FISH-positive percentage in those with residual tumor was significantly greater than in those without residual tumor (42.2% vs 17.6%, P = .003). Moreover, before and after the initial TUR, the percentage of conversion from FISH positive to FISH negative in those with residual tumor was significantly lower than in those without residual tumor (28.9% vs 58.9%, P < .001). No patients were observed with a FISH result of conversion from negative to positive in those with and without residual tumor after the initial TUR. FISH appears to be useful for the prediction of the presence of the residual tumor load after TUR of bladder UC.

Quantitative Fluorescence in Situ Hybridization and its Ability to Predict Bladder Cancer Recurrence and Progression to Muscle-Invasive Bladder Cancer

Fluorescence in situ hybridization (FISH) testing is used to detect bladder cancer in urine specimens. The purpose of this study was to determine whether there are associations between the percentage of chromosomally abnormal cells by FISH and time to bladder cancer recurrence and progression to metastasis. Clinical records were searched to identify patients with urine FISH results, history of non-invasive bladder cancer, and at least one follow-up pathological diagnosis. Covariates analyzed included age, gender, smoking status, treatment after FISH, cystoscopy result, and prior stage of bladder cancer. The percentage of abnormal cells (hazard ratio [HR] 1.03, 95% CI 1.02-1.03; P < 0.001), age (HR 1.02, 95% CI 1.00-1.03; P = 0.033), male gender (HR 0.60, 95% CI 0.41-0.87; P < 0.001), treatment (HR 0.37, 95% CI 0.25-0.55; P < 0.001), and history of TIS/T1-stage tumors (HR 1.66, 95% CI 1.23-2.24; P = 0.001) were significantly associated with time to cancer recurrence. Time to invasive cancer was significantly associated with the percentage of abnormal cells (HR 1.02, 95% CI 1.01, 1.03; P < 0.001), history of TIS/T1 tumor (HR 3.73, 95% CI 1.88, 7.38; P = 0.001), and treatment (HR 0.33, 95% CI 0.13, 0.83; P = 0.019), suggesting that the percentage of abnormal cells independently predicts cancer recurrence and progression to invasive disease in patients with a history of non-invasive bladder cancer.

Chromosomal abnormalities detected by multicolor fluorescence in situ hybridization in fine-needle aspirates from patients with small lymphocytic lymphoma are useful for predicting survival.

Fine-needle aspiration (FNA) of lymph nodes is commonly used to assess disease progression in patients with small lymphocytic lymphoma (SLL). Although cytologic features are helpful for diagnosing typical SLL and transformed large-cell lymphoma (tLCL), SLL in accelerated phase (SLLacc) is more difficult to diagnose. Additional tests are needed to identify those patients who are transforming to a higher-grade lymphoma. This study evaluated the use of a multicolor fluorescence in situ hybridization (FISH) probe panel specifically designed for chronic lymphocytic leukemia (CLL)/SLL and assessed the association between FISH findings and cytologic diagnosis, proliferation index, and risk of death. FNA specimens from 50 patients (32 men and 18 women; mean age, 57 years [range, 36-77 years]) with histologically confirmed CLL and/or SLL were evaluated in this study for chromosomal abnormalities of 11q22 (ATM), 12, 13q14.3, 13q34.3 (LAMP1), and 17p13.1 (p53) by using a multiprobe FISH kit. One of the 50 cases was excluded because of an insufficient number of cells for FISH analysis. The FISH findings were compared with the cytologic diagnoses (26 SLLs, 12 SLLaccs, and 11 tLCLs), Ki-67 immunostaining, and risk of death. Abnormal signal patterns for 17p13.1 and 13q34.3 were associated with tLCL. Aberrations of 17p13.1 were found to be significantly associated with Ki-67 staining. Of the 49 patients with interpretable FISH results, 22 (45%) had died at the time of the study, with a mean overall survival time of 17 months after FNA. Patients with aberrations of 17p13.1 and 11q22 had 3.7 and 2.7 times the risk of death, respectively, compared with patients with normal patterns. FISH can be performed on FNA specimens from patients with a history of SLL/CLL. Chromosomal aberrations of 17p13.1 and 11q22 are associated with an increased risk of death. Knowledge of genetic abnormalities from FNAs may be useful in deciding when and how to treat indolent or progressive SLL.

Screening of Subtelomeric Rearrangements in 100 Korean Pediatric Patients with Unexplained Mental Retardation and Anomalies Using Subtelomeric FISH (Fluorescence In Situ Hybridization)

Rearrangements of the subtelomeric regions of chromosomes account for a significant proportion of the underlying genetic defects in both idiopathic mental retardation (MR) and multiple congenital anomalies. To detect the rearrangements, a set of subtelomeric fluorescence in situ hybridization (FISH) probes has been developed. The aim of this study was to reveal the frequency of subtelomeric rearrangements in Korean patients with MR or multiple anomalies. We performed a FISH study using a commercially available subtelomeric FISH probes on a series of unrelated Korean pediatric patients with MR or multiple anomalies without identifiable causes. We used a checklist to evaluate the developmental delay and/or MR. Patients who were shown to have chromosome abnormalities, metabolic disorders, or recognizable dysmorphic syndromes by clinical and laboratory findings were excluded. As a result, 100 patients were eligible for the Subtelomeric FISH study, and a total of 29 patients (29%) were suspected to have subtelomeric rearrangements on initial screening by the multiprobe FISH kit. Among theses, confirmatory FISH studies by using single locus-specific FISH probes were performed in 24 patients. One patient (a 10-yr-old girl) was confirmed to have rearrangement, deletion of the telomeric portion of the short arm of chromosome 4 (4p). Her clinical manifestation was compatible with Wolf-Hirschhorn syndrome, which is known to be caused by 4p deletion. The frequency of subtelomeric rearrangements in this study was 1.1% (1/95), lower than those previously reported (0.5-16.3%). We suggest that subtelomeric FISH test is a useful screening tool for patients with idiopathic MR and/or dysmorphism regardless of its false positive value.

Utility of a multiprobe fluorescence in situ hybridization assay in the detection of superficial urothelial bladder cancer

We evaluated the performance of a multiprobe FISH (fluorescence in situ hybridization) assay for noninvasive detection of superficial urothelial carcinoma (UC) in the bladder, in comparison to urinary cytology. Voided urine samples from 74 patients with superficial UC were analyzed by both techniques. Urine samples from 19 patients with muscle-invasive tumors and from 19 healthy control subjects were also studied. For FISH analysis, labeled probes for chromosomes 3, 7, 9, and 17 were used to assess chromosomal abnormalities indicative of malignancy. We found a significant difference between the overall sensitivity of FISH and cytology in superficial UC detection (70.3 versus 35.1%, respectively; P < 0.0001). This significant difference was maintained when superficial UCs were broken down into low grade (52.8 versus 13.9%, respectively; P < 0.0005) and high grade (86.8 versus 55.3%, respectively; P < 0.0015) tumors. Overall specificity was 100% for cytology and 94.7% for FISH (difference not significant). Of patients with suspicious cytology, 69% were positive by FISH. Together, these findings suggest that FISH assay for chromosomes 3, 7, 9, and 17 has a higher sensitivity than cytology and a similar specificity in the detection of superficial UC—which could be useful for reducing some cystoscopies in the accurate follow-up usually performed in these patients.

Visualization of interphase chromosomes in postmitotic cells of the human brain by multicolour banding (MCB)

Molecular cytogenetics offers the unique possibility of investigating numerical and structural chromosomal aberrations in interphase nuclei of somatic cells. Previous fluorescence in-situ hybridization (FISH) investigations gave hints of numerical chromosomal imbalances in the human brain, present as low-level mosaicism. However, as precise identification of aneuploidy rates in somatic tissues faces major difficulties due to the limitations of FISH using whole chromosome painting or centromeric probes, in this study low-level mosaicism in the human brain was addressed for the first time using microdissection-based multicolour banding (MCB) probe sets. We demonstrated that MCB is suitable for this application and leads to more reliable results than the use of centromeric probes in parallel on the same samples. Autosomes and the active X chromosome appear as discrete metaphase chromosome-like structures, while the inactive X chromosome is condensed in more than 95% of interphase nuclei. The frequency of stochastic aneuploidy was found to be 0.2-0.5% (mean 0.35%) per autosome pair, 2% for the X chromosome in the female brain, and 0.4% in the male brain, giving a cumulative frequency of aneuploidy of approximately 10% in the adult brain. Moreover, MCB as well as multi-probe FISH using centromeric probes revealed associated signals in a large proportion of brain cells (10-40%). While co-localized signals could not be discriminated from numerical chromosome imbalances after FISH using centromeric probes, interphase MCB allows such differentiation. In summary, MCB is the only approach available at present that provides the possibility of characterizing the chromosomal integrity of arbitrary interphase cell populations. Thus, cytogenetics is no longer limited in its application to dividing cells, which is a great step forward for brain research.

MLPA vs multiprobe FISH: comparison of two methods for the screening of subtelomeric rearrangements in 50 patients with idiopathic mental retardation

Subtelomeric rearrangements not visible by conventional cytogenetic analysis have been reported to occur in approximately 5% of patients with unexplained mental retardation (MR). As the prevalence of MR is high, many patients need to be screened for these chromosomal abnormalities routinely. Multiplex ligation-dependent probe amplification (MLPA) is a new technique for measuring sequence dosage, allowing large number of samples to be processed simultaneously and thus significantly reducing laboratory work. We have assessed its performance for the detection of subtelomeric rearrangements by comparing the results with those of our previous multiprobe fluorescence in situ hybridization (FISH) assay. We have tested 50 patients with idiopathic MR, dysmorphic features, congenital malformations, and/or familial history of MR. Our results show a high degree of concordance between the two techniques for the 50 samples tested. On the basis of these results, we conclude that MLPA is a rapid, accurate, reliable, and cost-effective alternative to FISH for the screening of subtelomeric rearrangements in patients with idiopathic MR.

The value of fluorescence in situ hybridization in the diagnosis and prognosis of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. Prognosis is related to clinical staging and cytogenetic findings. Conventional cytogenetic analysis of CLL reveals abnormalities in approximately one third of patients. Fluorescence in situ hybridization (FISH) is analytically more sensitive than conventional cytogenetics for specific chromosomal abnormalities. To evaluate the usefulness of FISH in CLL, a study of 100 CLL patients comparing conventional cytogenetics and a commercially available multiprobe FISH kit was undertaken. One hundred consecutive CLL patients (67 males, 33 females) were studied. The male–female patient ratio was ∼2.0 to 1. Twenty-eight percent (28/98) of patients had abnormal karyotypes by conventional cytogenetics (one patient had no specimen for conventional cytogenetics and one had an unanalyzable karyotype), and of those 19/100 (19%) had more than one chromosomal abnormality. Sixty-four percent (64/100) of the patients were positive for at least one abnormality by the FISH probes used. The following abnormalities were noted with FISH: 11q22 ATM, 23/100 (23%); trisomy 12, 11/100 (11%); 13q14.3, 40/100 (40%); 13q34.3, 4/100 (4%); 17p13.1, 12/100 (12%). Conventional karyotypes revealed 2 patients with abnormalities of chromosome 6 (which FISH did not address); 11 with abnormalities of 11 or 11q; 6 with trisomy 12; and 4 with abnormalities of 17. Aberrations of 11q and 17p are reported to have a poor prognosis in CLL. FISH can identify abnormalities missed with conventional cytogenetics and is helpful in diagnosis, prognosis, and evaluation of therapy for CLL. Additional chromosomal changes are identified with conventional cytogenetics that are not addressed by the multiprobe FISH kit.

Multiprobe FISH for enhanced prediction of recurrent bladder cancer

The early detection of recurrent urothelial cancer is a great challenge for both urologists and pathologists. Cytology is a sensitive and highly specific tool for the diagnosis of high-grade bladder cancer and carcinoma in situ, but is not reliable in low-grade tumors. Therefore, cystoscopy has remained the gold standard for surveillance after resection of bladder cancer. Recent data suggest that multi-target fluorescence in situ hybridization (FISH) can markedly improve the sensitivity of urinary cytology. The goal of this study was to investigate the utility of the recently developed FISH assay Uro-Vysion for the prediction of recurrence during surveillance. 134 bladder washes obtained during a negative follow-up cystoscopy from 127 patients were analyzed. Positive cytology was strongly associated with subsequent recurrence, emphasizing the importance of conventional cytology for bladder cancer surveillance. A positive UroVysion FISH test significantly predicted recurrence when cases with rare tetraploic cells were considered as negative. Taken together, both cytology and FISH help to better determine the risk of bladder cancer recurrence in order to establish more individualized follow-up schemes.

Clinical Utility of a Multiprobe FISH Assay in Voided Urine Specimens for the Detection of Bladder Cancer and its Recurrences, Compared with Urinary Cytology

To evaluate the clinical utility of a Multi-color FISH (fluorescence in situ hybridization) assay in voided urine specimens for the detection of bladder cancer and its recurrences, comparing the results with those afforded by urinary cytology. Voided urine samples from 86 patients were obtained for urine cytology and FISH analysis. The latter was performed using a mixture of fluorescent labeled DNA probes for the centromeric regions of chromosomes 3, 7 and 17, and the 9p21 region. Cystoscopy with biopsy or tumor resection was performed in all patients, comparing the pathological results with the cytological and FISH findings. Urinary cytology affords an overall sensitivity of 63.8%, the figure being 25% for grade 1, 66.6% for grade 2 and 94.7% for grade 3 tumors. The sensitivities for FISH were 53.3% for grade 1, 83.3% for grade 2 and 100% for grade 3 tumors, with an overall sensitivity of 80.4%. The specificities of urinary cytology and FISH were 86.1 and 85.3%, respectively. FISH improves the sensitivity rates obtained with urine cytology for bladder cancer detection in all tumor grades and stages, and offers similar specificity. FISH doubles the accuracy of urinary cytology in application to low grade-stage tumors, and detects all high grade infiltrating tumors.

Integrated karyotyping of sorghum by in situ hybridization of landed BACs.

The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.

High incidence of chromosome 13 deletion in multiple myeloma detected by multiprobe interphase FISH

Multiple myeloma (MM) is a hypoproliferative malignancy yielding informative karyotypes in no more than 30% of newly diagnosed cases. Although cytogenetic and molecular deletion of chromosome 13 is associated with poor prognosis, a MM tumor suppressor gene (TSG) has not been identified. To localize a minimal deleted region of chromosome 13, clonotypic plasma cells from 50 consecutive patients with MM were subjected to interphase fluorescence in situ hybridization (FISH) analysis using a panel of 11 probes spanning the entire long arm of chromosome 13. Whereas chromosome 13 abnormalities were absent in plasma cells from 25 normal donors, 86% of patients with MM demonstrated such aberrations. Heterogeneity, both in deletion frequency and extent, was confirmed by simultaneous FISH with 2 chromosome 13 probes. Deletion hot spots were noted at D13S272 (70%) and D13S31 (64%), 2 unlinked loci at 13q14. Homozygous deletions at these loci occurred in 12% (simultaneously in 8%) of the cases. Molecular deletions were found in all 14 patients with morphologic deletions, in 21 of 24 with uninformative karyotypes, and 8 of 12 patients with karyotype abnormalities lacking chromosome 13 deletion. Homozygous deletion of any marker was noted in 4% with low and in 36% with higher plasma cell labeling index greater than 0.4% (P = .01). The absence of increasing deletion incidence and extent with therapy duration suggests that the observed lesions are not induced by treatment. The high incidence and extent of chromosome 13 deletions require the correlation of specific deletion(s) with poor prognosis. These analyses will provide valuable guidance toward cloning of an MM-TSG.

High incidence of chromosome 13 deletion in multiple myeloma detected by multiprobe interphase FISH

AbstractMultiple myeloma (MM) is a hypoproliferative malignancy yielding informative karyotypes in no more than 30% of newly diagnosed cases. Although cytogenetic and molecular deletion of chromosome 13 is associated with poor prognosis, a MM tumor suppressor gene (TSG) has not been identified. To localize a minimal deleted region of chromosome 13, clonotypic plasma cells from 50 consecutive patients with MM were subjected to interphase fluorescence in situ hybridization (FISH) analysis using a panel of 11 probes spanning the entire long arm of chromosome 13. Whereas chromosome 13 abnormalities were absent in plasma cells from 25 normal donors, 86% of patients with MM demonstrated such aberrations. Heterogeneity, both in deletion frequency and extent, was confirmed by simultaneous FISH with 2 chromosome 13 probes. Deletion hot spots were noted at D13S272 (70%) and D13S31 (64%), 2 unlinked loci at 13q14. Homozygous deletions at these loci occurred in 12% (simultaneously in 8%) of the cases. Molecular deletions were found in all 14 patients with morphologic deletions, in 21 of 24 with uninformative karyotypes, and 8 of 12 patients with karyotype abnormalities lacking chromosome 13 deletion. Homozygous deletion of any marker was noted in 4% with low and in 36% with higher plasma cell labeling index greater than 0.4% (P = .01). The absence of increasing deletion incidence and extent with therapy duration suggests that the observed lesions are not induced by treatment. The high incidence and extent of chromosome 13 deletions require the correlation of specific deletion(s) with poor prognosis. These analyses will provide valuable guidance toward cloning of an MM-TSG.