Amplification of the 3q chromosomal region as a specific marker in cervical cancer.

OBJECTIVE: Chromosome 3q gain has been consistently observed in cervical intraepithelial neoplasia grades 2 and 3 (CIN 2,3) and squamous cell carcinomas of the cervix. There are a number of potential clinical uses of testing for 3q gain in liquid cytology specimens, including the identification of subsets of women with atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion cytology who are at greatest risk of having CIN 2,3 and would thus benefit most from immediate colposcopy. The objective of this study was to establish the sensitivity and specificity of 3q gain for discriminating between CIN 2,3 and normal. STUDY DESIGN: Residual cytology specimens were collected from 199 women. Liquid-based cytology (LBC) was used for the selection of subjects, with women with high-grade squamous intraepithelial lesion or high-grade squamous intraepithelial lesion who had colposcopy and adjudicated biopsy-confirmed CIN 2,3 forming the disease-positive group (n = 28) and women doubly negative for both cytology and high-risk human papillomavirus (hrHPV) testing forming the disease-negative group (n = 171). A single slide was prepared from each residual LBC specimen and analyzed for 3q gain by fluorescent in situ hybridization, using a probe specific for the 3q26 region and a control probe for the chromosome 7 centromere. Two approaches were compared for the determination of 3q gain. The first was based on the analysis of an entire cervical cytology slide for the presence of rare cells with a high copy number (>4 copies) for the 3q locus. The second approach was based on the analysis of 400 cells to determine the percentage with 3 or more copies of the 3q locus. RESULTS: Using the approach based on the detection of rare cells with a high copy number (>4 copies) for the 3q locus, 26 of the specimens from women with CIN 2,3 and none of the 171 specimens from women who were both hrHPV and cytology negative was positive for 3q gain. This translates to a sensitivity of 92.9% (95% confidence interval [CI], 76.5-98.9%), a specificity of 100% (95% CI, 97.8-100%), a positive predictive value of 100% (95% CI, 86.7-100%), and a negative predictive value of 98.8% (95% CI, 95.9-99.8), for distinguishing CIN 2,3 from normal. CONCLUSION: These data support the potential clinical use of 3q gain for the evaluation of women in a number of clinical situations, including women with atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesion, and those who are hrHPV positive.

Is there a gain in chromosome 3q in the pathway to anal cancer?

BACKGROUND: Chromosome 3q gain has been identified in human papillomavirus-infected cervical cancer cells. OBJECTIVE: We sought to identify the presence of chromosomal 3q gain in anal neoplasia. DESIGN: This was a retrospective cohort. SETTINGS: The study was conducted in a group colorectal surgery practice. PATIENTS: Fifty-two patients with no dysplasia, low-grade dysplasia, high-grade dysplasia, or anal cancer were studied. INTERVENTIONS: Pairs of biopsy specimens were paraffin embedded and reviewed. One of each slide pair was stained with hematoxylin and eosin and the second processed for fluorescence in situ hybridization. The hybridized set was deparaffinized first and then hybridized with a probe for the chromosome 3q26 region. Then, slides were scanned using an automated fluorescence microscopy system that analyzed defined areas of the tissue to enumerate all of the nuclei for hybridized probe signals to detect chromosome 3q gain. MAIN OUTCOME MEASURES: We measured for gain in chromosome 3q26. RESULTS: We identified chromosome 3q gain in 7 (78%) of 9 patients with squamous-cell cancer, 8 (53%) of 15 high-grade dysplasia samples, 0 of 12 low-grade dysplasia samples, and 0 of 16 samples with no dysplasia. The sensitivity for high-grade or invasive neoplasia was 58%, with a specificity of 100%. The positive predictive value of the test was 100% for detecting high-grade dysplasia and/or squamous-cell cancer from no dysplasia, and the negative predictive value of the test was 62%. LIMITATIONS: This study was limited by its small sample size and retrospective design. CONCLUSIONS: Chromosome 3q gain represents an important shared pathway to tumorigenesis in cervical and anal neoplasia. Multiple potential diagnostic roles exist for this easily performed test in the evaluation of anal neoplasia.

3q26 amplification is an effective negative triage test for LSIL: a historical prospective study.

BACKGROUND: Women with low grade squamous intraepithelial lesions (LSIL) at cervical cancer screening are currently referred for further diagnostic work up despite 80% having no precancerous lesion. The primary purpose of this study is to measure the test characteristics of 3q26 chromosome gain (3q26 gain) as a host marker of carcinogenesis in women with LSIL. A negative triage test may allow these women to be followed by cytology alone without immediate referral to colposcopy. METHODS AND FINDINGS: A historical prospective study was designed to measure 3q26 gain from the archived liquid cytology specimens diagnosed as LSIL among women attending colposcopy between 2007 and 2009. 3q26 gain was assessed on the index liquid sample; and sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were measured at immediate triage and at 6-16 months after colposcopic biopsy. The sensitivity of 3q26 gain measured at immediate triage from automated and manually reviewed tests in 65 non-pregnant unique women was 70% (95% CI: 35, 93) with a NPV of 89% (95% CI: 78, 96). The sensitivity and NPV increased to 80% (95% CI: 28, 99) and 98% (95% CI: 87, 100), respectively, when only the automated method of detecting 3q26 gain was used. CONCLUSIONS: 3q26 gain demonstrates high sensitivity and NPV as a negative triage test for women with LSIL, allowing possible guideline changes to routine surveillance instead of immediate colposcopy. Prospective studies are ongoing to establish the sensitivity, specificity, PPV and NPV of 3q26 gain for LSIL over time.

Role of chromosome 3q26 gain in predicting progression of cervical dysplasia.

OBJECTIVE: This study aimed to determine whether 3q26 gain can predict which low-grade squamous intraepithelial lesions (LSILs) and atypical squamous cells of undetermined significance (ASCUSs) will progress to higher-grade squamous intraepithelial lesion (HSIL). METHODS: Liquid cytology specimens of LSIL and ASCUS from 73 women were examined using fluorescent in situ hybridization (FISH) for the detection of 3q26 gain. All women underwent colposcopy and biopsy at the initial visit and 40 of them with histology showing cervical intraepithelial neoplasia 1 (CIN 1) or human papillomavirus infection (koilocytosis) were included in the study. They were reevaluated with liquid cytology, colposcopy, and biopsy after a median follow-up of 17.5 months. RESULTS: A total of 40 cases were analyzed (31 LSILs and 9 ASCUSs). Of these cases, 8 (20%; 6 LSILs and 2 ASCUSs) were positive and 32 (80%) were negative for 3q26 gain according to FISH. Three of the 8 positive women (38%) progressed to HSIL/CIN 2 or worse, whereas none of the 32 negative women did so. 3q26 gain could predict progression with a negative predictive value of 100% (95% confidence interval, 89.1%-100%). In addition, women positive for 3q26 gain had a significantly lower regression rate compared with negative women (P = 0.009). CONCLUSIONS: In this first prospective study, 3q26 gain in LSIL/ASCUS cytology exhibited an impressive negative predictive value for progression to HSIL/CIN 2 or worse. Thus, 3q26 gain may be useful in stratifying patients' risk for progression and possibly alter management and reduce cost of follow-up.

Efficiency of manual scanning in recovering rare cellular events identified by fluorescence in situ hybridization: simulation of the detection of fetal cells in maternal blood.

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1-10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.

Determination of HER2 gene status by fully automated fluorescence microscopy.

OBJECTIVE: To examine fluorescence in situ hybridization (FISH) in HER2 amplification in response rates to trastuzumab therapy and both taxane and anthracycline-based chemotherapy regimens. STUDY DESIGN: A total of 400 tumor sections were analyzed over an 8-month period. The sections were hybridized with probes for the HER2 gene and chromosome 17 centromere using standard FISH methods and analyzed on an automated fluorescence microscopy system. RESULTS: Reliable and valid methods for identification of the patients that will respond to treatment with trastuzumab are needed in order to achieve maximum therapy efficacy and maintain cost efficiency. FISH-based analysis is potentially an objective and reproducible approach to determination of HER2 gene status; however, manual FISH counting is a laborious task and subject to inter and intraobserver variability. CONCLUSION: The system described in this paper is a valuable tool in providing a consistent approach to the interpretation of breast tumor tissue analyzed by FISH analysis. In addition to consistency, an automated system provides a record of the images produced that can be of immediate benefit in multiple review of a difficult or equivocal case and long-term benefit in terms of providing a permanent case record.

Digitized microscopy in the diagnosis of bladder cancer: analysis of >3000 cases during a 7-month period.

BACKGROUND: Fluorescent in situ hybridization (FISH) analysis of urine samples has proven to be a valuable adjunctive test to urine cytology for both diagnosis and monitoring recurrence of urothelial carcinoma. Automated FISH analysis has the potential to improve laboratory efficiency and to reduce interobserver and intraobserver variability, resulting in more accurate, reproducible, assay performance. METHODS: A total of 3200 slides containing urine specimens, hybridized with the UroVysion Bladder Cancer Kit (Abbott Molecular, Des Plaines, Illinois), a 4-probe set for chromosomes 3, 7, 17, and 9p21, was evaluated at Acupath Laboratories. The slides were analyzed over a 7-month period, using the Ikoniscope - oncoFISH bladder Test System (Ikonisys, New Haven, Connecticut). RESULTS: Analysis included the incorporation of a "flagging" system developed by Acupath Laboratories to identify cases, based on specific criteria, likely to benefit from further manual review. By using US Food and Drug Administration (FDA)-cleared scoring criteria, 96.3% of the slides could be reported directly from the automated scan, requiring no manual review of the slide. For the remaining 3.7% of the samples (all of which were very hypocellular), a manual review of each slide subsequently allowed diagnoses to be successfully reported. The average scan time was 31.7 minutes, and the average slide scan review time was 8.3 minutes. CONCLUSIONS: This study demonstrated the value of an automated approach to the analysis of FISH slides, affording the benefit of high-throughput while providing the user with the necessary images and tools to quickly and accurately report a case.

Prenatal diagnosis of trisomy 21 through detection of trophoblasts in cervical smears.

BACKGROUND: Fetal cells exfoliate in the uterine cavity during early pregnancy and are a potential source of material for NIPD. AIMS: This study was designed to test the hypothesis that fetal cells obtained from the uterine cervix during the first trimester of pregnancy could be utilized for prenatal diagnosis of chromosomal aneuploidy. STUDY DESIGN: Fetal cells retrieved from the distal endocervical canal during the first trimester of pregnancy were hybridized with chromosome 21 specific FISH probes and analyzed with an automated fluorescence microscope. SUBJECTS AND OUTCOME MEASURES: Cells with 3 copies of chromosome 21 were detected in 5 out of 5 trisomy 21 pregnancies. RESULTS: The number of trisomic cells detected ranged from 1 to 27 with a median value of 5. CONCLUSIONS: FISH-based scanning can identify trisomy 21 pregnancies by analysis of routine cervical brushings. The approach offers the potential for non-invasive prenatal diagnosis as early as 5 weeks gestation.

Amplification of the chromosome 3q26 region shows high negative predictive value for nonmalignant transformation of LSIL cytologic finding.

OBJECTIVE: The chromosome 3q26 region is a biomarker for cervical cancer. Women with low-grade squamous intraepithelial lesions (LSIL) currently are referred for immediate colposcopy. The objective of this study was to determine the negative predictive value of the 3q26 amplification test for the persistence or regression of LSIL. STUDY DESIGN: Archival thin layer cytologic slides of 47 women (14-67 years old) with LSIL were linked to histologic and cytologic end points. To determine 3q status, the slides were hybridized for the chromosome 3q26 region and for the centromere of chromosome 7, as a control, with the use of the standard fluorescent in situ hybridization methods. RESULTS: The negative predictive value of 3q26 gain for the development of cervical intraepithelial neoplasia grade 2/3 within 1 year was 93% (95% confidence interval, 68- 100); after 21 months, its negative predictive value was 100% (95% confidence interval, 29-100). CONCLUSION: The 3q26 gain might help identify women with LSIL who do not need colposcopy.

Gain of 3q26: a genetic marker in low-grade squamous intraepithelial lesions (LSIL) of the uterine cervix.

OBJECTIVE: Physicians have few resources for determining which LSIL will progress to HSIL or regress. Recently the chromosome 3q26 region was found to be amplified in patients with cervical cancer. The frequency of this 3q gain increased with severity of dysplasia. The primary objective of this study was to evaluate an automated FISH assay for detection of 3q gain in liquid cytology samples as a potential tool for risk stratification and triaging. METHODS: Slides prepared from 257 liquid cytology specimens (97 Negative, 135 LSIL 25 HSIL) were hybridized with a single-copy probe for the chromosome 3q26 region and a probe for the centromeric alpha-repeat sequence of chromosome 7, using standard FISH methods. Using automated analysis, the total number of nuclei and the number of nuclei with >2 signals for 3q26 were determined, using a 20x objective. The nuclei were rank ordered based on number of 3q26 FISH signals. The 800 nuclei with the highest number of signals were scored using both FISH probes and nuclei with increased numbers of 3q signals were enumerated. RESULTS AND CONCLUSIONS: Analysis of 257 specimens demonstrated that a fully automated FISH scoring system can detect 3q gain in liquid cytology samples. A fully automated method for determination of 3q gain in liquid cytology may be the assay necessary to implement routine testing. Additional studies to validate the utility of this technology are needed.

Detection of circulating fetal cells utilizing automated microscopy: potential for noninvasive prenatal diagnosis of chromosomal aneuploidies.

OBJECTIVE: As fetal cells can be indisputably identified through detection of Y FISH signals, we utilized an automated microscopy system developed to identify and enumerate cells bearing X and Y FISH signals. We further investigated the potential of fetal hemoglobin expression as a gender independent marker for automated identification of fetal cells. METHOD: For FISH-based scanning, verified fetal cells were identified based on the presence of a single X-signal and individual signals for each of the two Y FISH probes. For cell identification based on fetal hemoglobin expression, putative fetal cells were verified based on the presence of signals for anti-gamma or anti-epsilon globin antibody, and FISH signals for the X- and Y- chromosomes. RESULTS: Fetal cells were identified, by FISH-based scanning, in 28 of the 29 maternal samples from pregnancies with male fetuses. Simple density gradient centrifugation achieved a 3- to 5-fold increase in the number of fetal cells detected. CONCLUSION: Automated microscopy identified fetal cells in both first and second trimester maternal blood samples. Although we were unable to detect fetal erythroblasts in numbers sufficient for clinical diagnosis, the ability to reliably detect fetal cells by FISH-based scanning opens the possibility for prenatal detection of chromosomal aberrations utilizing circulating fetal cells.

Fully automated FISH examination of amniotic fluid cells.

OBJECTIVE: Fluorescence in situ hybridization (FISH) analysis has become a valuable adjunct in cytogenetics, providing a rapid screen for common chromosome abnormalities that is particularly helpful in prenatal diagnosis. FISH analysis using standard microscopy is expensive and labor intensive, requiring both a high skill level and subjective signal interpretation. A reliable fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. METHODS: The efficacy of an automated system was compared to standard manual FISH analysis. Two sets of slides were generated from each of 152 amniotic fluid samples. Following hybridization with a standard panel of five chromosome FISH probes, one set of slides was evaluated using manual microscopy. The other set was evaluated using an automated microscopy system. RESULTS: A diagnostic outcome was obtained for all 152 samples using manual microscopy and for 146 of 152 (96%) samples using automated microscopy. Three cases of aneuploidy were detected. For those samples for which a diagnostic outcome was determined by both manual and automated microscopy, 100% concordance was observed. All FISH analysis results were confirmed by karyotype. CONCLUSION: These data suggest that an automated microscopy system is capable of providing accurate and rapid enumeration of FISH signals in amniocytes.

Automated microscopy of amniotic fluid cells: detection of FISH signals using the FastFISH imaging system.

OBJECTIVE: FISH (fluorescence in situ hybridization) analysis is a valuable adjunct to cytogenetics that provides a rapid screen for common abnormalities. However, FISH is expensive, labor-intensive, and requires a high skill level and subjective signal interpretation. A fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. METHODS: In this study we blindly compared automated FISH signal acquisition and display against standard FISH analysis. A total of 62 amniocentesis samples were prepared using the AneuVysion multicolor DNA probe kit and probed for chromosomes 13, 18, 21, X, and Y. Two sets of slides were produced from each sample. Fifty cells were scored in each slide. One set was evaluated using standard manual microscopy and the other using the automated image acquisition and display capabilities of the Ikoniscope fastFISH amnio Test System. This system uses epifluorescence optics, along with optimized slide management to process slides automatically. RESULTS: A 100% concordance was observed between the results obtained using manual microscopy and the automated system. There was also 100% concordance between the FISH results and those obtained by conventional karyotyping. CONCLUSION: Our data suggest that the automated system is capable of providing accurate and rapid identification and display of cells and FISH signals.

The expanding panorama of split hand foot malformation.

The split hand/foot malformation is a developmental defect of the extremities resulting from errors in the initiation and maintenance of the apical ectodermal ridge. The phenotype is genetically heterogeneous, and it can be identified either as an isolated phenotypic manifestation or as a constituent component of a malformation syndrome. This overview describes the clinical phenotype, related animal models, and the evolving genetic heterogeneity of the malformation.

Endogenously expressed multimeric self-cleaving hammerhead ribozymes ablate mutant collagen in cellulo.

Hammerhead ribozymes are small catalytic RNA molecules that can be targeted to any RNA molecule containing a putative cleavage site. We developed a vector (pCOLZ) that uses the COL1A1 promoter to drive expression of a self-cleaving multimeric ribozyme (M8Rz547) and its monomeric counterpart (Rz547). The ribozymes were stably coexpressed in MC3T3-E1 osteoblasts expressing a truncated COL1A1 target transcript. The multimeric ribozyme exhibited self-cleavage to derivative fragments, including monomers. Increased expression of ribozymes was found in cells expressing the multimeric ribozyme. A modest reduction of truncated target transcript and protein was seen in cells expressing the ribozyme monomer, while nearly complete ablation of target transcript and protein occurred in cells expressing the ribozyme multimer. A reversion to a more normal collagen phenotype, measured as an increase in fibril diameter and restored fibrillar architecture, and a decreased rate of collagen turnover were seen in cells expressing the ribozyme multimer.

Automated detection of rare fetal cells in maternal blood: eliminating the false-positive XY signals in XX pregnancies.

OBJECTIVE: The purpose of this study was to develop a new method to help differentiate XX from XY signals in maternal blood from women carrying XY fetuses. STUDY DESIGN: We have developed a system to scan automatically for cells that bear X and Y fluorescence in situ hybridization signals. These XY target cells are identified by scans at low (x20) magnification, and all identified targets are revisited and verified at high (x100) magnification. The viewer software component of the system displays x20 images of all cells and intracellular fluorescence in situ hybridization signals that are present in each of the 4000 optical fields per slide, along with x100 images of automatically detected target cells. RESULTS: We initially examined 36,000 fields from 18 slides in 12 pregnancies (6 male and 6 female) using our system that is based on fluorescence in situ hybridization with a single probe for the X-chromosome and a single probe for the Y-chromosome and found XY nuclei in all samples, regardless of fetal gender. In the second phase of the study, a refinement of the approach that incorporated 2 independent probes for the Y-chromosome resulted in a false-positive rate for detection of XY nuclei in XX cases <0.00005%. CONCLUSION: Our data suggest that this system may allow for excellent "signal to noise" separation, which is required absolutely for fetal cell methods to differentiate aneuploid from normal pregnancies. Quantitation of fetal cells in the maternal circulation and standardization of processes that have been developed for their enrichment are crucial to moving fetal cell assessment from esoteric basic science to applied new technology.

Haplotype analysis enables the diagnosis of Marfan syndrome.

Marfan syndrome is an inherited disorder of connective tissue associated with aneurysmal rupture of the ascending aorta. Timely and accurate diagnosis has reduced the mortality and morbidity associated with this disorder through expectant observation and implementation of appropriate prophylactic therapy. To this end, haplotype analysis using polymorphic genetic markers in close proximity to the Fibrillin-1 gene (FBN1) were employed to aid in the diagnosis of two individuals who did not meet the clinical diagnostic criteria.

A recurring FBN1 gene mutation in neonatal Marfan syndrome.

BACKGROUND: Marfan syndrome is an autosomal dominant disorder of connective tissue caused by mutations in the fibrillin 1 gene (FBN1). FBN1 mutations have been associated with a broad spectrum of phenotypes. Neonatal Marfan syndrome has unique clinical manifestations and mutations. OBJECTIVE: To determine if there is a discernible genotypic-phenotypic correlation associated with the unique mutation in neonatal Marfan syndrome. STUDY DESIGN: A newborn exhibited many typical characteristics of neonatal Marfan syndrome, including arachnodactyly; contractures of both elbows, knees, and ankles; small-joint laxity; dilated cardiomyopathy; valvular dysplasia and insufficiency; congestive heart failure; and pulmonary emphysema. Three atypical features were also discovered: a right diaphragmatic hernia, a myocardial mass, and left main-stem bronchomalacia. She died at 3(1/2) months of age. Total RNA was extracted from skin fibroblasts and amplified by means of reverse transcriptase polymerase chain reaction amplification with FBN1-specific primers. The complementary DNA fragments were sequenced. RESULTS: A single T-to-C transition at nucleotide 3276 (T3276C) was identified and confirmed at the DNA level by sequencing of genomic DNA. This results in a substitution of threonine for isoleucine. CONCLUSIONS: Neonatal Marfan syndrome is a unique clinical entity with recurring mutation hot spots in exons 24 to 27 and 31 to 32 of the FBN1 gene. Some clinical features in this case report are unusual for neonatal Marfan syndrome. This is the third report of this T3276C mutation in the FBN1 gene with unusual clinical manifestations. We conclude that there is a genotypic-phenotypic correlation associated with this mutation.

Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene.

The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor.