The use of TMA for interlaboratory validation of FISH testing for detection of HER2 gene amplification in breast cancer.

HER2 fluorescence in situ hybridization (FISH) testing for breast cancer is largely limited to academic centers and commercial laboratories. As testing demands increase, methods for rapid and cost-effective technical validation and quality assessment will be required. Tissue microarray (TMA), a technique for high-throughput biomarker evaluation, could help facilitate these needs. Our objective was to assess the usefulness of TMA technology for validation of HER2 FISH testing. Two TMA blocks containing paired cores from 41 breast cancers were constructed. HER2 FISH was performed in parallel at two institutions and the results compared. One institution, with considerable HER2 FISH experience, served as the reference laboratory. HER2 chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) were compared to the FISH results. For positive and negative results, the concordance rate between laboratories was 100%. Using kappa statistical analysis to determine interobserver agreement, HER2 to chromosome 17 gene copy ratios showed strong agreement between laboratories with kappa = 0.85 (perfect agreement = 1.0). Four cases displaying low-level amplification by CISH contained chromosome 17 polysomy and gene copy ratios of <2.0 by FISH. Good concordance was observed between HER2 IHC and in situ hybridization testing. TMA is a robust and effective method for the technical validation of HER2 FISH testing and should be considered for use by quality assessment programs.     

The specificity of interphase FISH translocation probes in formalin fixed paraffin embedded tissue sections is readily assessed using automated staining and scoring of tissue microarrays constructed from murine xenografts

Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.     

Fully Automated Fluorescent in situ Hybridization (FISH) Staining and Digital Analysis of HER2 in Breast Cancer: A Validation Study

HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH) procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with supervised automated analysis with the Visia imaging D-Sight digital imaging platform. HER2 assessment was performed on 328 formalin-fixed/paraffin-embedded invasive breast cancer tumors on tissue microarrays (TMA) and 100 (50 selected IHC 2+ and 50 random IHC scores) full-sized slides of resections/biopsies obtained for diagnostic purposes previously. For digital analysis slides were pre-screened at 20x and 100x magnification for all fluorescent signals and supervised-automated scoring was performed on at least two pictures (in total at least 20 nuclei were counted) with the D-Sight HER2 FISH analysis module by two observers independently. Results were compared to data obtained previously with the manual Abbott FISH test. The overall agreement with Abbott FISH data among TMA samples and 50 selected IHC 2+ cases was 98.8% (κ = 0.94) and 93.8% (κ = 0.88), respectively. The results of 50 additionally tested unselected IHC cases were concordant with previously obtained IHC and/or FISH data. The combination of the Leica FISH system with the D-Sight digital imaging platform is a feasible method for HER2 assessment in routine clinical practice for patients with invasive breast cancer.     

Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia

Genomic aberrations have increasingly gained attention as prognostic markers in B-cell chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) has improved the detection rate of genomic alterations in CLL from approximately 50% using conventional cytogenetics to greater than 80%. More recently, array comparative genomic hybridization (CGH) has gained popularity as a clinical tool that can be applied to detect genomic gains and losses of prognostic importance in CLL. Array CGH and FISH are particularly useful in CLL because genomic gains and losses are key events with both biologic and prognostic significance, while balanced translocations have limited prognostic value. Although FISH has a higher technical sensitivity, it requires separate, targeted hybridizations for the detection of alterations at genomic loci of interest. Array CGH, on the other hand, has the ability to provide a genome-wide survey of genomic aberrations with a single hybridization reaction. Array CGH is expanding the known genomic regions of importance in CLL and allows these regions to be evaluated in the context of a genome-wide perspective. Ongoing clinical trials are evaluating the use of genomic aberrations as tools for risk-stratifying patients for therapy, thus increasing the need for reliable and high-yield methods to detect these genomic changes. In this review, we consider the use of array CGH as a clinical tool for the identification of genomic alterations with prognostic significance in CLL, and suggest ways to integrate this test into the clinical molecular diagnostic laboratory work flow.     

Dual-Color Fluorescence In Situ Hybridization Reveals an Association of Chromosome 8q22 but Not 8p21 Imbalance with High Grade Invasive Breast Carcinoma

We previously reported molecular karyotype analysis of invasive breast tumour core needle biopsies by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) (Walker et al, Genes Chromosomes Cancer, 2008 May;47(5):405-17). That study identified frequently recurring gains and losses involving chromosome bands 8q22 and 8p21, respectively. Moreover, these data highlighted an association between 8q22 gain and typically aggressive grade 3 tumors. Here we validate and extend our previous investigations through FISH analysis of tumor touch imprints prepared from excised breast tumor specimens. Compared to post-surgical tumor excisions, core needle biopsies are known to be histologically less precise when predicting tumor grade. Therefore investigating these chromosomal aberrations in tumor samples that offer more reliable pathological assessment is likely to give a better overall indication of association. A series of 60 breast tumors were screened for genomic copy number changes at 8q22 and 8p21 by dual-color FISH. Results confirm previous findings that 8p loss (39%) and 8q gain (74%) occur frequently in invasive breast cancer. Both absolute quantification of 8q22 gain across the sample cohort, and a separate relative assessment by 8q22:8p21 copy number ratio, showed that the incidence of 8q22 gain significantly increased with grade (p = 0.004, absolute and p = 0.02, relative). In contrast, no association was found between 8p21 loss and tumor grade. These findings support the notion that 8q22 is a region of interest for invasive breast cancer pathogenesis, potentially harboring one or more genes that, when amplified, precipitate the molecular events that define high tumor grade.     

Comparison of in situ hybridization methods for the assessment of HER-2/neu gene amplification status in breast cancer using a tissue microarray

BackgroundThis project compared HER-2/neu gene status in breast cancers, as demonstrated by FISH (fluorescent in situ hybridization) and CISH (chromogenic in situ hybridization) and using a tissue microarray (TMA). The study also aimed to show whether the TMA technique could be used in clinical diagnostics, rather than remain a scientific tool.Materials and methodsA TMA was constructed using 121 breast cancer specimens, 6 cores from each specimen. Demonstration and assessment of HER-2/neu gene status was by FISH (Vysis Path) and CISH (DAKO Duo CISH).ResultsThe 121 breast cancer specimens were divided into 3 groups by HER-2 status, as determined by immunohistochemistry. In the HER-2 negative group no amplification was observed in 36 out of 40 cases. 3 cases showed amplification by both methods and one by CISH alone. The equivocal HER-2 group showed no amplification in 30 out of 41 cases and amplification in 9 cases. One case was FISH negative CISH positive and one was discarded. In the HER-2 positive group, amplification was confirmed in 37 of the 40 cases by both methods. 3 cases were unsuitable for assessment.ConclusionsThis study indicated that CISH is a sensitive alternative to FISH in detecting HER2 gene amplification and may replace FISH in HER2 testing. Good agreement was observed between methods (98.5% – 119 out of 121 cases).Furthermore, as only 4 out of 121 cases were unsuitable for assessment (no signal or missing TMA cores) – it may be feasible to use TMA in diagnostics.     

Improved procedure for fluorescence in situ hybridization on tissue microarrays

BACKGROUND: The recently developed tissue microarray (TMA) technology allows the arrangement of up to a thousand tissue specimens on a single microscope slide. This technology enables researchers to perform gene copy number studies on very large series of archival formalin-fixed tissues using fluorescence in situ hybridization (FISH). However, the hybridization properties of individual archival specimens can vary considerably. Therefore a highly optimized protocol is needed to fulfill the task of producing evaluable hybridization signals simultaneously in hundreds of specimens in a TMA. METHODS: The performance of two different FISH protocols, the standard protocol for paraffin embedded tissues and our new optimized protocol, was tested on TMAs using probes for the HER-2 and ZNF217 genes as well as the chromosome 17 centromere. RESULTS: The new protocol resulted in greatly increased signal intensity and an almost 30% increase in the number of tissue samples with evaluable hybridization signals. CONCLUSIONS: Our improved protocol for FISH on TMAs provides standardized hybridization conditions leading to high-quality hybridization signals in the majority of specimens. The increases in the signal intensity and the number of evaluable samples are extremely important for the successful analyses of TMAs by FISH and will allow the utilization of the TMA technology in its full potential.     

Genome-wide examination of chromosomal aberrations in neuroblastoma SH-SY5Y cells by array-based comparative genomic hybridization

Most neuroblastoma cells have chromosomal aberrations such as gains, losses, amplifications and deletions of DNA. Conventional approaches like fluorescence in situ hybridization (FISH) or metaphase comparative genomic hybridization (CGH) can detect chromosomal aberrations, but their resolution is low. In this study we used array-based comparative genomic hybridization to identify the chromosomal aberrations in human neuroblastoma SH-SY5Y cells. The DNA microarray consisting of 4000 bacterial artificial chromosome (BAC) clones was able to detect chromosomal regions with aberrations. The SH-SY5Y cells showed chromosomal gains in 1q12 approximately q44 (Chr1:142188905-246084832), 7 (over the whole chromosome), 2p25.3 approximately p16.3 (Chr2:18179-47899074), and 17q 21.32 approximately q25.3 (Chr17:42153031-78607159), while chromosomal losses detected were the distal deletion of 1p36.33 (Chr1:552910-563807), 14q21.1 approximately q21.3 (Chr14:37666271- 47282550), and 22q13.1 approximately q13.2 (Chr22:36885764-4190 7123). Except for the gain in 17q21 and the loss in 1p36, the other regions of gain or loss in SH-SY5Y cells were newly identified.     

PROBER: oligonucleotide FISH probe design software

PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. AVAILABILITY: http://prober.cshl.edu     

Structural unbalanced chromosome rearrangements resolved by comparative genomic hybridization.

The identification of unbalanced structural chromosome rearrangements using conventional cytogenetic techniques depends on recognition of the unknown material from its banding pattern. Even with optimally banded chromosomes, when large chromosome segments are involved, cytogeneticists may not always be able to determine the origin of extrachromosomal material and supernumerary chromosomes. We report here on the application of comparative genomic hybridization (CGH), a new molecular-cytogenetic assay capable of detecting chromosomal gains and losses, to six clinical samples suspected of harboring unbalanced structural chromosome abnormalities. CGH provided essential information on the nature of the unbalanced aberration investigated in five of the six samples. This approach has proved its ability to resolve complex karyotypes and to provide information when metaphase chromosomes are not available. In cases where metaphase chromosome spreads were available, confirmation of CGH results was easily obtained by fluorescence in situ hybridization (FISH) using specific probes. Thus the combined use of CGH and FISH provided an efficient method for resolving the origin of aberrant chromosomal material unidentified by conventional cytogenetic analysis.     

Frequent gains on chromosome arms 1q and/or 8q in human endometrial cancer

Comparative genomic hybridization (CGH) was employed to survey genomic regions with increased and decreased copy number of the DNA sequence in 15 endometrial cancers [10 cases with microsatellite instability positive (MI+) and 5 cases with MI–]. Twelve of these 15 tumors (80%) showed abnormalities in copy number at one or more of the chromosomal regions. There were no regions with frequent chromosomal losses. Conversely, 11 of 15 cases (73%) showed gains on chromosome arms 1q (8/15; 53%) and/or 8q (6/15; 40%). Concordant gains of both chromosome arms 1q and 8q were observed in all three endometrial cancers of histological grade 3. These results suggest that these two chromosomal regions may contain genes whose increased expression contributes to development and/or progression of endometrial carcinogenesis. Two cases were further analyzed by fluorescence in situ hybridization (FISH) using three probes on chromosome 1 and two probes on chromosome 8 to more accurately determine increases in copy number. We found gains of chromosome 1q to 2.9–3.6 copies per cell and on 8q to 4.4 copies per cell. Received: 9 March 1997 / Accepted: 2 June 1997     

Development of bioinformatics resources for display and analysis of copy number and other structural variants in the human genome

The discovery of an abundance of copy number variants (CNVs; gains and losses of DNA sequences >1 kb) and other structural variants in the human genome is influencing the way research and diagnostic analyses are being designed and interpreted. As such, comprehensive databases with the most relevant information will be critical to fully understand the results and have impact in a diverse range of disciplines ranging from molecular biology to clinical genetics. Here, we describe the development of bioinformatics resources to facilitate these studies. The Database of Genomic Variants (http://projects.tcag.ca/variation/) is a comprehensive catalogue of structural variation in the human genome. The database currently contains 1,267 regions reported to contain copy number variation or inversions in apparently healthy human cases. We describe the current contents of the database and how it can serve as a resource for interpretation of array comparative genomic hybridization (array CGH) and other DNA copy imbalance data. We also present the structure of the database, which was built using a new data modeling methodology termed Cross-Referenced Tables (XRT). This is a generic and easy-to-use platform, which is strong in handling textual data and complex relationships. Web-based presentation tools have been built allowing publication of XRT data to the web immediately along with rapid sharing of files with other databases and genome browsers. We also describe a novel tool named eFISH (electronic fluorescence in situ hybridization) (http://projects.tcag.ca/efish/), a BLAST-based program that was developed to facilitate the choice of appropriate clones for FISH and CGH experiments, as well as interpretation of results in which genomic DNA probes are used in hybridization-based experiments.     

Comparative genomic hybridization arrays in clinical pathology: progress and challenges

Array-based comparative genomic hybridization (array CGH) genome scanning is a powerful method for the global detection of gains and losses of genetic material in both congenital and neoplastic disorders. When used as a clinical diagnostic test, array CGH combines the whole genome perspective of traditional G-banded cytogenetics with the targeted identification of cryptic chromosomal abnormalities characteristic of fluorescence in situ hybridization (FISH). However, the presence of structural variants in the human genome can complicate analysis of patient samples, and array CGH does not provide morphologic information about chromosome structure, balanced translocations, or the actual chromosomal location of segmental duplications. Identification of such anomalies has significant diagnostic and prognostic implications for the patient. We therefore propose that array CGH should be used as a guide to the presence of genomic structural rearrangements in germline and tumor genomes that can then be further characterized by FISH or G-banding, depending on the clinical scenario. In this article, we share some of our experiences with diagnostic array CGH and discuss recent progress and challenges involved with the integration of array CGH into clinical laboratory medicine.     

A strategy combining flow sorting and comparative genomic hybridization for studying genetic aberrations at different stages of colorectal tumorigenesis in ulcerative colitis

BACKGROUND: DNA aneuploidy has been shown to increase the risk of developing dysplasia in ulcerative colitis (UC) and is related to tumorigenesis in the colorectum. Therefore, it is of particular interest to study genetic aberrations behind DNA aneuploidization during colorectal carcinogenesis. We wanted to elucidate further the relationship between mucosal morphology and DNA aberrations in UC. METHODS: DNA flow cytometry was applied to multiple lesions including regenerative, dysplastic, and carcinomatous mucosa from the colectomy specimen of a male patient with long-standing UC. The lesions harbored multiple DNA aneuploid stemlines that were subjected to flow sorting. We analyzed gene alterations by degenerate oligonucleotide primer (DOP; universal primers) polymerase chain reaction (PCR)-based comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) in diploid and aneuploid sorted cells. RESULTS: DOP-PCR-based CGH shows gains and losses that can be verified by FISH. We show that with this approach one can study genetic evolution of distinct DNA diploid and aberrant subpopulations through defined stages of colorectal tumorigenesis. This includes getting information related to tumor heterogeneity that cannot be obtained by CGH with DNA extracted from nonsorted cell populations. Genetic imbalance was also detected in diploid nondysplastic flow-sorted mucosal cells from the same bowel. CONCLUSIONS: Similar gains and losses were found in aneuploid dysplasias and carcinomas at widely separated locations in the same bowel, indicating a common selection pressure in different areas of the same bowel. The common aberrations may be of importance for progression from dysplasia to carcinoma.     

Mosaikbefunde in der Microarray-Diagnostik bei prä- und postnatalen Untersuchungen

Molecular karyotyping of array-based genomic hybridization (microarrays) not only detects copy number variations, genomic gains and losses but also provides the assessment of specific mosaicism. This article gives an overview on parameters influencing the detection of mosaicism and different cases with mosaicism detected by comparative genomic hybridization (CGH) arrays and by single nucleotide polymorphism (SNP) arrays. Furthermore, a possibility is provided to calculate the percentage of a given mosaicism.     

Effects of chromosomal variations on pharmacokinetic activity of zolpidem in healthy volunteers: an array-based comparative genomic hybridization study

Zolpidem has been known as a very safe and effective hypnotic drug used to treat a variety of patients with insomnia. Even though the same dose of the medicine is administered to each patient, the blood level of zolpidem and the time required to obtain peak concentration are not consistent among different people. We evaluated the relationship between the peak concentrations of zolpidem and chromosomal imbalances using a high-resolution genome-wide array-based comparative genomic hybridization (CGH) in 16 healthy volunteers in order to detect the genetic factors underlying the variations. The present study showed that chromosomal losses were detected in the 4q35.2, 9p13.1 and 9p12 regions, and those gains were indicated in the 2p14, 11q13.4 and 15q11.2 regions. The abnormal regions were confirmed by fluorescence in situ hybridization (FISH) and real-time PCR. It is suggested that array-CGH analysis may be used as a measure for pharmacogenomic applications in the patients with insomnia and for further exploration of candidate genomic regions implicated in sleep disturbances.     

Discordant results obtained for different methods of HER-2/neu testing in breast cancer--a question of standardization, automation and timing

BACKGROUND: HER-2/neu positivity is required for the selection of stage IV breast cancer patients for trastuzumab therapy. We compared the results of the recommended immunohistochemistry (IHC) evaluation with the automated ACIS IHC system and with fluorescence in situ hybridization (FISH). These HER-2/neu tissue results were correlated with the serum HER-2/neu (sHER-2/neu) levels at the time of metastatic spread. PATIENTS AND METHODS: A total of 61 IHC slides from 30 patients were stained using the HercepTest. HER-2/neu gene amplification was determined using the Ventana FISH assay. sHER-2/neu levels were measured with the Oncogene Science" ELISA kit. The concordance of HER-2/neu results was determined using the concordance index Kappa (kappa). RESULTS: The best concordance between any IHC and FISH was found for the automated ACIS system (88.5%, kappa=0.68, category "good"). The comparison between the manual interpretations and the automated IHC was categorized as "very good" (95.1%, kappa=0.85). The median sHER-2/neu level of FISH positive patients was significantly higher (67 ng/mL) than that of FISH negative patients (17 ng/mL, p=0.018). The increase in HER-2/neu positivity comparing tissue to stage IV serum was statistically significant (p=0.001). CONCLUSIONS: The concordance between conventional IHC and computerized analysis was very good. The number of patients with stage IV breast cancer with an elevated sHER-2/neu level was much higher than HER-2/neu positivity in tissue. This discrepancy is only partially explained by the influence of tumor load. Patients with an elevated sHER-2/neu level and no tissue overexpression should be considered for retesting of tissue or a new biopsy.     

Adjusting Breast Cancer Patient Prognosis with Non-HER2-Gene Patterns on Chromosome 17

Background

HER2 and TOP2A gene status are assessed for diagnostic and research purposes in breast cancer with fluorescence in situ hybridization (FISH). However, FISH probes do not target only the annotated gene, while chromosome 17 (chr17) is among the most unstable chromosomes in breast cancer. Here we asked whether the status of specifically targeted genes on chr17 might help in refining prognosis of early high-risk breast cancer patients.

Methods

Copy numbers (CN) for 14 genes on chr17, 4 of which were within and 10 outside the core HER2 amplicon (HER2- and non-HER2-genes, respectively) were assessed with qPCR in 485 paraffin-embedded tumor tissue samples from breast cancer patients treated with adjuvant chemotherapy in the frame of two randomized phase III trials.

Principal Findings

HER2-genes CN strongly correlated to each other (Spearman’s rho >0.6) and were concordant with FISH HER2 status (Kappa 0.6697 for ERBB2 CN). TOP2A CN were not concordant with TOP2A FISH status (Kappa 0.1154). CN hierarchical clustering revealed distinct patterns of gains, losses and complex alterations in HER2- and non-HER2-genes associated with IHC4 breast cancer subtypes. Upon multivariate analysis, non-HER2-gene gains independently predicted for shorter disease-free survival (DFS) and overall survival (OS) in patients with triple-negative cancer, as compared to luminal and HER2-positive tumors (interaction p = 0.007 for DFS and p = 0.011 for OS). Similarly, non-HER2-gene gains were associated with worse prognosis in patients who had undergone breast-conserving surgery as compared to modified radical mastectomy (p = 0.004 for both DFS and OS). Non-HER2-gene losses were unfavorable prognosticators in patients with 1–3 metastatic nodes, as compared to those with 4 or more nodes (p = 0.017 for DFS and p = 0.001 for OS).

Conclusions

TOP2A FISH and qPCR may not identify the same pathology on chr17q. Non-HER2 chr17 CN patterns may further predict outcome in breast cancer patients with known favorable and unfavorable prognosis.     

Comparative analysis of comparative genomic hybridization microarray technologies: report of a workshop sponsored by the Wellcome Trust

BACKGROUND: Array-comparative genomic hybridization (CGH), although providing much higher resolution compared with conventional CGH, has not yet become a widely applied method for the analysis of genomic gains and losses. METHODS: In January 2002, the Wellcome Trust sponsored a workshop where many of the laboratories developing this technology met to compare different methodologies for array-CGH. Fourteen groups participated, comprising 11 from Europe and 3 from the United States. To facilitate objective analysis, each laboratory constructed arrays using the same anonymous clones and performed a series of test hybridizations using identical genomic DNAs. RESULTS: A figure of merit (FM) was developed to summarize entire collections of data from each laboratory in a single measurement. The FMs consistently showed that a few groups produced quantitative array hybridization data of high quality, whereas a majority achieved a lower standard. CONCLUSIONS: The conclusions of the workshop were that polymerase chain reaction-based methods for the amplification of large insert clones for arraying were effective for array-CGH. It was also concluded that hybridizations performed under coverslips or in automated hybridization apparatus were less effective than hybridizations performed in simple wells with gentle rocking. A common experience by the participants was the batch-to-batch variability of commercial Cot1 preparations in their ability to suppress hybridization to repeat sequences. (Supplementary material for this article can be found in the online issue, which is available at http://www.interscience.wiley.com/jpages/0196-4763/suppmat/49_2/v49.43.html or at http://www.sanger.ac.uk/HGP/Cytogenetics/Publications/Cytometry Sept 2002/Supplemental.pdf.)