Conventional histological and cytological staining with simultaneous immunohistochemistry enabled by invisible chromogens.

Conventional histological stains, such as hematoxylin plus eosin (H&E), and immunohistochemistry (IHC) are mainstays of histology that provide complementary diagnostic information. H&E and IHC currently require separate slides, because the stains would otherwise obscure one another. This consumes small specimen, limiting the total amount of testing. Additionally, performing H&E and IHC on different slides does not permit comparison of staining at the single cell level, since the same cells are not present on each slide, and alignment of tissue features can be problematic due to changes in tissue landscape with sectioning. We have solved these problems by performing conventional staining and IHC on the same slide using invisible IHC chromogens, such that the chromogens are not visible when viewing the conventional stain and the conventional stain is excluded from images of the IHC. Covalently deposited chromogens provided a convenient route to invisible chromogen design and are stable to reagents used in conventional staining. A dual-camera brightfield microscope system was developed that permits simultaneous viewing of both visible conventional stains and invisible IHC chromogens. Simultaneous staining was demonstrated on several formalin-fixed paraffin-embedded tissue specimens using single and duplex IHC, with chromogens that absorb ultraviolet and near infrared light, followed by H&E staining. The concept was extended to other conventional stains, including mucicarmine special stain and Papanicoulou stain, and further extended to cytology specimens. In addition to interactive video review, images were recorded using multispectral imaging and image processing to provide flexible production of color composite images and enable quantitative analysis.

Brightfield multiplex immunohistochemistry with multispectral imaging.

Brightfield microscopy is the preferred method of pathologists for diagnosing solid tumors, utilizing common staining techniques such as hematoxylin and eosin staining and immunohistochemistry (IHC). However, as our understanding of the complex tumor microenvironment grows, there is increasing demand for multiplexed biomarker detection. Currently, multiplexed IHC assays are almost exclusively based on immunofluorescence because brightfield techniques are limited by the broad spectral absorption of chromogens and a reliance on conventional 3-channel color cameras. In this work, we overcome these limitations by combining new chromogens possessing narrow absorbance bands with matched illumination channels and monochrome imaging. Multiplex IHC was performed using four or five covalently deposited chromogens and hematoxylin nuclear stain to preserve morphological context and detail. Brightfield illumination was provided with a tungsten lamp/filter wheel combination or filtered light emitting diodes to provide up to 12 illumination wavelengths. In addition, an automated rapid imaging system was developed, using a synchronized 12-LED illuminator, that could capture images at all wavelengths in under 1 s. In one example, a four-biomarker multiplex assay was designed and used to distinguish regions of adenocarcinoma and squamous cell carcinoma in non-small cell lung cancer. The technology was also validated with a five-biomarker assay in prostate cancer. Spectrally unmixed images of each biomarker demonstrated concordant expression patterns with DAB single stain on serial sections, indicating faithful identification of each biomarker. In each assay, all chromogens were well resolved by spectral unmixing to remove spectral crosstalk. While further characterization and refinement of the assay, and improvements in automation and user interface are necessary for pathologist acceptance, this approach to multiplex IHC and multispectral imaging has the potential to accelerate adoption of multiplexing by combining the medical value of high-order multiplexing with the speed, pathologist familiarity, and broadly established clinical utility of brightfield microscopy.

Chromogenic immunohistochemical quadruplex provides accurate diagnostic differentiation of non-small cell lung cancer.

Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung malignancies, the vast majority of which are non-small cell lung carcinomas (NSCLC). Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or by immunohistochemistry, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung. Each marker is represented by a distinct color that can be read by a pathologist, using standard, bright field microscopy. We evaluated the ability of pathologists to differentiate NSCLCs using the multiplexed assay as compared to standard, single marker per slide diaminobenzidine (DAB)-based IHC. All cases in a cohort of 264 NSCLCs showed concordance of information (including positivity of stain, intensity of stain and coverage) between single IHC stains and the multiplex assay. This new multiplex IHC offers the capability to accurately diagnose and sub-classify primary lung NSCLCs, while conserving precious tissue for additional testing.

Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies.

The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.

Covalently deposited dyes: a new chromogen paradigm that facilitates analysis of multiple biomarkers in situ.

Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated. Studies reported herein demonstrate utility of tyramine-chemistry for synthesis of a wide variety of new chromogenic dye conjugates useful for multiplexed in situ analysis using conventional light microscopes. The dyes, useful individually or in blends to generate new colors, provide signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. It is anticipated that this new paradigm will enable generation of a wide variety of new chromogens, useful for both research and clinical biomarker analysis that will benefit clinicians and patients.

A Multicenter Study of a Fluorescence In Situ Hybridization Probe Set for Diagnosing High-Grade Dysplasia and Adenocarcinoma in Barrett's Esophagus.

BACKGROUND AND AIMS: Preliminary single-institution data suggest that fluorescence in situ hybridization (FISH) may be useful for detecting high-grade dysplasia (HGD) and esophageal adenocarcinoma (EA) in patients with Barrett's esophagus (BE). This multicenter study aims to validate the measurement of polysomy (gain of at least two loci) by FISH as a way to discriminate degrees of dysplasia in BE specimens. METHODS: Tissue specimens were collected from four different hospitals and read by both the local pathology department ("Site diagnosis") and a single central pathologist ("Review diagnosis") at a separate institution. The specimens then underwent FISH analysis using probes 8q24 (MYC), 9p21 (CDKN2A), 17q12 (ERBB2), and 20q13 (ZNF217) for comparison. A total of 46 non-BE, 42 non-dysplastic specialized intestinal metaplasia (SIM), 23 indefinite-grade dysplasia (IGD), 10 low-grade dysplasia (LGD), 29 HGD, and 42 EA specimens were analyzed. RESULTS: We found that polysomy, as detected by FISH, was the predominant chromosomal abnormality present as dysplasia increased. Polysomy was also the best predictor for the presence of dysplasia or EA when comparing its area under the curve to that of other FISH abnormalities. We observed that if at least 10% of cells had polysomy within a specimen, the FISH probe was able to differentiate between EA/HGD and the remaining pathologies with a sensitivity of 80% and a specificity of 88%. CONCLUSIONS: This study demonstrates that using FISH to determine the percentage of cells with polysomy can accurately and objectively aid in the diagnosis of HGD/EA in BE specimens.

MET and PTEN gene copy numbers and Ki-67 protein expression associate with pathologic complete response in ERBB2-positive breast carcinoma patients treated with neoadjuvant trastuzumab-based therapy.

BACKGROUND: Pathologic complete response (pCR) after neoadjuvant chemotherapy for breast cancer is associated with improved prognosis in aggressive tumor subtypes, including ERBB2- positive tumors. Recent adoption of pCR as a surrogate endpoint for clinical trials in early stage breast cancer in the neoadjuvant setting highlights the need for biomarkers that, alone or in combination, help predict the likelihood of response to treatment. METHODS: Biopsy specimens from 29 patients with invasive ductal carcinoma treated with trastuzumab-based therapy prior to definitive resection and pathologic staging were evaluated by dual color bright field in situ hybridization (dual ISH) using probes for MET, TOP2A, PTEN, and PIK3CA genes, each paired with centromeric probes to their respective chromosomes (chromosomes 7, 17, 10, and 3). Ki-67 expression was assessed by immunohistochemistry (IHC). Various parameters describing copy number alterations were evaluated for each gene and centromere probe to identify the optimal parameters for clinical relevance. Combinations of ISH parameters and IHC expression for Ki-67 were also evaluated. RESULTS: Of the four genes and their respective chromosomes evaluated by ISH, two gene copy number parameters provided statistically significant associations with pCR: MET gain or loss relative to chromosome 7 (AUC = 0.791, sensitivity = 92 % and specificity = 67 % at optimal cutoff, p = 0.0032) and gain of PTEN (AUC = 0.674, sensitivity = 38 % and specificity = 100 % at optimal cutoff, p = 0.039). Ki-67 expression was also found to associate significantly with pCR (AUC = 0.726, sensitivity = 100 % and specificity = 42 % at optimal cutoff, p = 0.0098). Combining gain or loss of MET relative to chromosome 7 with Ki-67 expression further improved the association with pCR (AUC = 0.847, sensitivity = 92 % and specificity = 83 % at optimal cutoffs, p = 0.0006). CONCLUSIONS: An immunogenotypic signature of low complexity comprising MET relative copy number and Ki-67 expression generated by dual ISH and IHC may help predict pCR in ERBB2-positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab. These findings require validation in additional patient cohorts.

Identification of FISH biomarkers to detect chromosome abnormalities associated with prostate adenocarcinoma in tumour and field effect environment.

BACKGROUND: To reduce sampling error associated with cancer detection in prostate needle biopsies, we explored the possibility of using fluorescence in situ hybridisation (FISH) to detect chromosomal abnormalities in the histologically benign prostate tissue from patients with adenocarcinoma of prostate. METHODS: Tumour specimens from 33 radical prostatectomy (RP) cases, histologically benign tissue from 17 of the 33 RP cases, and 26 benign prostatic hyperplasia (BPH) control cases were evaluated with Locus Specific Identifier (LSI) probes MYC (8q24), LPL (8p21.22), and PTEN (10q23), as well as with centromere enumerator probes CEP8, CEP10, and CEP7. A distribution of FISH signals in the tumour and histologically benign adjacent tissue was compared to that in BPH specimens using receiver operating characteristic curve analysis. RESULTS: The combination of MYC gain, CEP8 Abnormal, PTEN loss or chromosome 7 aneusomy was positive in the tumour area of all of the 33 specimens from patients with adenocarcinomas, and in 88% of adjacent histologically benign regions (15 out of 17) but in only 15% (4 out of 26) of the benign prostatic hyperplasia control specimens. CONCLUSIONS: A panel of FISH markers may allow detection of genomic abnormalities that associate with adenocarcinoma in the field adjacent to and surrounding the tumour, and thus could potentially indicate the presence of cancer in the specimen even if the cancer focus itself was missed by biopsy and histology review.

Fluorescence in situ hybridization markers for prediction of cervical lymph node metastases.

The presence of lymph node metastases is associated with poor prognosis in early stage cervical cancer. As of yet, no molecular markers predicting lymph node metastases have been identified. We examined single genetic markers and a composite marker, comprised of three fluorescence in situ hybridization (FISH) probes targeting the genes LAMP3, PROX1, and PRKAA1, in pretreatment cervical biopsies from 16 lymph node positive cases and 15 lymph node negative controls from women with stage IB and IIA cervical cancer. In addition, we determined clonal patterns by including CCND1 to compare the clonal constitution of primary tumors and associated lymph node metastases. The composite FISH marker allowed for classification of patients into those with and without lymph node metastases with a sensitivity and specificity of 75% and 87%, respectively (P = 0.001). The positive predictive value and negative predictive value were 86% and 76%, respectively. Clonal patterns varied among the tumors. In many cases, changes between the primary tumor and lymph node metastases in the most common clones may indicate that certain clones have a growth advantage for establishing metastases in lymph nodes. We conclude that the composite FISH marker may be useful for determining risk for subsequent development of lymph node metastases in patients with cervical cancer.

Analysis of genetic copy number changes in cervical disease progression.

BACKGROUND: Cervical dysplasia and tumorigenesis have been linked with numerous chromosomal aberrations. The goal of this study was to evaluate 35 genomic regions associated with cervical disease and to select those which were found to have the highest frequency of aberration for use as probes in fluorescent in-situ hybridization. METHODS: The frequency of gains and losses using fluorescence in-situ hybridization were assessed in these 35 regions on 30 paraffin-embedded cervical biopsy specimens. Based on this assessment, 6 candidate fluorescently labeled probes (8q24, Xp22, 20q13, 3p14, 3q26, CEP15) were selected for additional testing on a set of 106 cervical biopsy specimens diagnosed as Normal, CIN1, CIN2, CIN3, and SCC. The data were analyzed on the basis of signal mean, % change of signal mean between histological categories, and % positivity. RESULTS: The study revealed that the chromosomal regions with the highest frequency of copy number gains and highest combined sensitivity and specificity in high-grade cervical disease were 8q24 and 3q26. The cytological application of these two probes was then evaluated on 118 ThinPrep samples diagnosed as Normal, ASCUS, LSIL, HSIL and Cancer to determine utility as a tool for less invasive screening. Using gains of either 8q24 or 3q26 as a positivity criterion yielded specificity (Normal +LSIL+ASCUS) of 81.0% and sensitivity (HSIL+Cancer) of 92.3% based on a threshold of 4 positive cells. CONCLUSIONS: The application of a FISH assay comprised of chromosomal probes 8q24 and 3q26 to cervical cytology specimens confirms the positive correlation between increasing dysplasia and copy gains and shows promise as a marker in cervical disease progression.

Basic principles of fluorescence and energy transfer.

Fluorescence is highly sensitive to environment, and the distance separating fluorophores and quencher molecules can provide the basis for effective homogeneous nucleic acid hybridization assays. Molecular interactions leading to fluorescence quenching include collisions, ground state and excited state complex formation, and long-range dipole-coupled energy transfer. These processes are well understood and equations are provided for estimating the effects of each process on fluorescence intensity. Estimates for the fluorescein-tetramethylrhodamine donor-acceptor pair reveal the relative contributions of dipole-coupled energy transfer, collisional quenching, and static quenching in several common assay formats, and illustrate that the degree of quenching is dependent upon the hybridization complex formed and the manner of label attachment.

The development of a fluorescence in situ hybridization assay for the detection of dysplasia and adenocarcinoma in Barrett's esophagus.

The goal of this study was to identify a set of fluorescence in situ hybridization probes for the detection of dysplasia and adenocarcinoma in patients with Barrett's esophagus. We examined 170 brushing specimens from 138 patients with Barrett's esophagus or a history of Barrett's esophagus using fluorescence in situ hybridization with probes to 5p15, 5q21-22, centromere 7, 7p12, 8q24.12-13, centromere 9, 9p21, centromere 17, 17p13.1, 17q11.2-12, 20q13.2, and centromere Y. Receiver-operator curves were used to determine the sensitivity and specificity of various four-probe combinations for detecting low-grade dysplasia, high-grade dysplasia, and esophageal adenocarcinoma. Endoscopic biopsy results were used as the gold standard. Numerous four-probe combinations provided a similarly high sensitivity and specificity. Of these, a set consisting of probes to 8q24, 9p21, 17q11.2, and 20q13.2 was found to have a sensitivity and specificity, respectively, of 70% and 89% for low-grade dysplasia, 84% and 93% for high-grade dysplasia, and 94% and 93% for esophageal adenocarcinoma. This probe set was chosen for future prospective clinical evaluations based on its high sensitivity and specificity, its ability to distinguish adenocarcinoma and high-grade or low-grade dysplasia from lesser diagnostic categories, and the favorable signal quality for each of the probes.

Cyclin E expression is a significant predictor of survival in advanced, suboptimally debulked ovarian epithelial cancers: a Gynecologic Oncology Group study.

Cyclin E is a key regulator of the G(1)-S transition. Abnormalities in cyclin E expression have been related to survival in a variety of cancers. This study evaluated the prognostic relevance of cyclin E in human ovarian cancer. Immunohistochemical expression of cyclin E was evaluated in 139 advanced, suboptimally debulked epithelial ovarian cancer specimens from patients treated on Gynecologic Oncology Group protocol 111. High cyclin E protein expression (> or =40% cyclin E positive tumor cells) was seen in 62 (45%) of the advanced, suboptimally debulked ovarian cancer patients. Expression of cyclin E was not associated with age, race, stage, grade, cell type, or amount of residual disease. High verses low cyclin E expression was associated with a shorter median survival (29 +/- 2 versus 35 +/- 3 months) and worse overall survival (P < 0.05). Univariate and multivariate regression analyses revealed that high relative to low cyclin E was associated with a 40-50% increase in the risk of death (hazard rate, P < or = 0.05). Fluorescence in situ hybridization was used in a subset of 20 cases to examine cyclin E gene amplification. Eight of 10 cases with high cyclin E expression exhibited amplification of the cyclin E gene, whereas only 1 of 10 cases with low expression displayed gene amplification (P < 0.006). High cyclin E expression was an independent poor prognostic factor for patients with advanced ovarian cancer, and it was associated with amplification of the cyclin E gene.

Detection of chromosomal instability in paired breast surgery and ductal lavage specimens by interphase fluorescence in situ hybridization.

PURPOSE: Ductal lavage is a new modality for collecting exfoliated breast cells with the goal of detecting early neoplasia. The purpose of our study was to evaluate the correlation between cancer-associated abnormalities in breast lesions and exfoliated breast cells collected by ductal lavage. EXPERIMENTAL DESIGN: We performed histopathologic, cytologic, and molecular cytogenetic analyses on 39 paired cases of surgically excised breast lesions and ductal lavage specimens collected immediately before surgery. RESULTS: Abnormal cytology was detected in 7 of 15 (47%) of the evaluable lavages collected from malignant cases, versus 4 of 19 (21%) of the evaluable lavages harvested from benign cases for a sensitivity and specificity of 47 and 79%, respectively. Interphase fluorescence in situ hybridization analysis of all evaluable lavages revealed numeric changes on chromosomes 1, 8, 11, and/or 17 in 10 of 14 (71%) specimens from malignant cases versus 2 of 18 (11%) from benign cases for a sensitivity and specificity of 71 and 89%, respectively. CONCLUSIONS: Our study demonstrates that cytologic and genetic abnormalities associated with breast cancer progression can be detected in ductal lavage cells collected from women with in situ and invasive breast cancer and suggests that fluorescence in situ hybridization may have superior sensitivity and specificity compared with conventional cytology.

Aberrant EGFR and chromosome 7 associate with outcome in laryngeal cancer.

OBJECTIVES/HYPOTHESIS: Epidermal growth factor receptor (EGFR) over-expression has been reported as a prognostic indicator in laryngeal cancer; however, the association with disease outcome has been inconsistent among studies. Here, we use fluorescence in situ hybridization (FISH) in addition to immunohistochemistry to assess laryngeal squamous cell carcinoma (SCC) to determine whether FISH can better predict patient outcome. STUDY DESIGN: Retrospective study on 59 patients presenting with advanced disease. METHODS: EGFR and chromosome 7 genomic statuses were measured using FISH, and EGFR expression was assessed by immunohistochemistry on formalin-fixed, paraffin-embedded specimens and correlated with outcome in the 59 patients. RESULTS: EGFR expression was marginally associated with outcome, whereas both EGFR and chromosome 7 FISH status were significantly associated with outcome, and the combination of EGFR and chromosome 7 FISH status provided the strongest association of any two combined parameters (P = .0004). Combining EGFR expression with EGFR and chromosome 7 FISH status provided further improvement (P > .0001). CONCLUSIONS: Measurements of EGFR and chromosome 7 FISH status, and to a lesser extent EGFR expression, have potential value in treatment planning for patients with laryngeal SCC.

Chromosomal abnormalities in non-small cell lung carcinomas and in bronchial epithelia of high-risk smokers detected by multi-target interphase fluorescence in situ hybridization.

Human lung carcinogenesis is accompanied by complex chromosomal changes that may be detected in interphase cells by fluorescence in situ hybridization (FISH) assay using recently developed multitarget DNA probes. Touch preparations of 20 non-small cell lung carcinomas, sputum specimens from 3 patients with lung cancer and from 11 ex-smokers without lung cancer, and cultured benign bronchial epithelium of 42 high-risk smokers, 9 of whom had concurrent invasive carcinoma, were tested using a four-color FISH probe (LAVysion) targeting centromere 6, 5p15.2, 7p12 (EGFR), and 8q24 (MYC). Significantly high frequencies of abnormal cells were found in each of the 20 NSCLC (100%) and in the 3 sputum specimens from lung cancer patients. None of the cytologically normal sputa contained FISH abnormalities. Cultured bronchial epithelial cells from 11 of 42 patients (26%) were abnormal for at least one probe. Abnormal FISH patterns had no association with gender, presence of tumor or histology. Multicolor FISH can readily detect chromosomal abnormalities in imprints and sputa from lung carcinomas. Chromosomal aneusomy is also frequent in bronchial epithelial cells from long-term smokers. The prognostic significance of the multicolor LAVysion FISH probe set should be validated in a controlled clinical trial.

Basic principles of fluorescence and energy transfer applied to real-time PCR.

Fluorescence is highly sensitive to environment, and the distance separating fluorophores and quencher molecules can provide the basis for effective homogeneous nucleic acid hybridization assays. Molecular interactions leading to fluorescence quenching include collisions, ground state and excited state complex formation, and long-range dipole-coupled energy transfer. These processes are well understood and equations are provided for estimating the effects of each process on fluorescence intensity. Estimates for the fluorescein-tetramethylrhodamine donor-acceptor pair reveal the relative contributions of dipole-coupled energy transfer, collisional quenching, and static quenching in several common assay formats, and illustrate that the degree of quenching is dependent upon the hybridization complex formed and the manner of label attachment.

Fluorescence in situ hybridization (FISH) as an ancillary diagnostic tool in the diagnosis of melanoma.

Although the clinical and pathologic diagnosis of some melanomas is clear-cut, there are many histopathologic simulators of melanoma that pose problems. Over-diagnosis of melanoma can lead to inappropriate therapy and psychologic burdens, whereas under-diagnosis can lead to inadequate treatment of a deadly cancer. We used existing data on DNA copy number alterations in melanoma to assemble panels of fluorescence in situ hybridization (FISH) probes suitable for the analysis of paraffin-embedded tissue. Using FISH data from a training set of 301 tumors, we established a discriminatory algorithm and validated it on an independent set of 169 unequivocal nevi and melanomas as well as 27 cases with ambiguous pathology, for which we had long-term follow-up data. An algorithm-using signal counts from a combination of 4 probes targeting chromosome 6p25, 6 centromere, 6q23, and 11q13 provided the highest diagnostic discrimination. This algorithm correctly classified melanoma with 86.7% sensitivity and 95.4% specificity in the validation cohort. The test also correctly identified as melanoma all 6 of 6 cases with ambiguous pathology that later metastasized. There was a significant difference in the metastasis free survival between test-positive and negative cases with ambiguous pathology (P=0.003). Sufficient chromosomal alterations are present in melanoma that a limited panel of FISH probes can distinguish most melanomas from most nevi, providing useful diagnostic information in cases that cannot be classified reliably by current methods. As a diagnostic aid to traditional histologic evaluation, this assay can have significant clinical impact and improve classification of melanocytic neoplasms with conflicting morphologic criteria.