Pathwork Tissue Of Origin Test Research Articles

A Multicenter Study Directly Comparing the Diagnostic Accuracy of Gene Expression Profiling and Immunohistochemistry for Primary Site Identification in Metastatic Tumors

Metastatic tumors with an uncertain primary site can be a difficult clinical problem. In tens of thousands of patients every year, no confident diagnosis is ever issued, making standard-of-care treatment impossible. Gene expression profiling (GEP) tests currently available to analyze these difficult-to-diagnose tumors have never been directly compared with the diagnostic standard of care, immunochemistry (IHC). This prospectively conducted, blinded, multicenter study compares the diagnostic accuracy of GEP with IHC in identifying the primary site of 157 formalin-fixed paraffin-embedded specimens from metastatic tumors with known primaries, representing the 15 tissues on the GEP test panel. Four pathologists rendered diagnoses by selecting from 84 stains in 2 rounds. GEP was performed using the Pathwork Tissue of Origin Test. Overall, GEP accurately identified 89% of specimens, compared with 83% accuracy using IHC (P=0.013). In the subset of 33 poorly differentiated and undifferentiated carcinomas, GEP accuracy exceeded that of IHC (91% to 71%, P=0.023). In specimens for which pathologists rendered their final diagnosis with a single round of stains, both IHC and GEP exceeded 90% accuracy. However, when the diagnosis required a second round, IHC significantly underperformed GEP (67% to 83%, P<0.001). GEP has been validated as accurate in diagnosing the primary site in metastatic tumors. The Pathwork Tissue of Origin Test used in this study was significantly more accurate than IHC when used to identify the primary site, with the most pronounced superiority observed in specimens that required a second round of stains and in poorly differentiated and undifferentiated metastatic carcinomas.

A prospective registry study assessing decision impact and patient outcomes following gene-expression profiling for tumor-site origin.

e22172 Background: A retrospective registry study has shown that gene-expression profiling (GEP) of the tissue of origin (Pathwork Tissue of Origin Test) of metastatic or poorly differentiated cancer changes the working diagnosis and management for the majority of patients. The FDA-cleared GEP test determines similarity of tumors of uncertain origin to gene expression patterns representing 60 morphologies in 15 cancer types. Inability to identify cancer primary sites presents challenges for patient treatment, outcomes, and prognosis. This study aims to prospectively assess impact of GEP on physician decisions and patient outcomes. Methods: A prospective, observational study is examining GEP’s impact on treatment changes and outcomes in adult patients with difficult-to-diagnose cancers. The study is assessing changes in diagnosis, management, oncologist confidence and perception of the test’s clinical utility, survival, therapy response, and other outcomes over 2 years of follow-up. Results before and after GEP are presented here for 18 patients. The study is designed to enroll 300 patients overall. Results: The primary working diagnosis changed for 37% of patients: 4 whose physicians obtained a working diagnosis while previously not having a tissue-specific diagnosis, and 3 for whom the primary diagnosis changed. After GEP, all patients had a working diagnosis. At least one aspect of management plans changed for 65% patients after GEP. Chemotherapy was ordered for 2 more patients and the regimen changed for 3 others; surgery was performed for 4 fewer, radiation for 1 fewer, and hospice for 2 more patients. After obtaining results from GEP, 58% of the treating oncologists “agreed” or “strongly agreed” that they were confident in both their diagnosis and treatment. Seventy-two percent believed that their changes in management would lead to improvement in at least one outcome (survival, response rate, time to relapse, quality of life, or patient anxiety). Conclusions: Current results of this prospective study are consistent with results of the prior retrospective study: GEP significantly changed and improved physician confidence in their diagnosis and treatment. Clinical trial information: NCT01649453.

Comparison of histopathology to gene expression profiling for the diagnosis of metastatic cancer

BackgroundDetermining the primary site of metastatic cancer with confidence can be challenging. Pathologists commonly use a battery of immunohistochemical (IHC) stains to determine the primary site. Gene expression profiling (GEP) has found increasing use, particularly in the most difficult cases. In this pilot study, a direct comparison between GEP and IHC-guided methods was performed.MethodsTen archived formalin-fixed paraffin embedded metastatic tumor samples for which the primary site had been clinically determined were selected. Five pathologists who were blinded to the diagnosis were asked to determine the primary site using IHC and other stains selected from a panel of 84 stains. Each pathologist was provided patient sex, biopsy site and gross sample description only. Slides were digitized using ScanScope®XT at 0.25 μm/pixel. Each evaluating pathologist was allowed to provide a diagnosis in three stages: initial (after reviewing the H&E image), intermediate (after reviewing images from the first batch of stains) and final diagnosis (after the second batch of stains if requested). GEP was performed using the only FDA-cleared test for this intended use, the Pathwork Tissue of Origin Test. No sample information was provided for GEP testing except for patient sex. Results were reported as the tumor tissue type with the highest similarity score.ResultsIn this feasibility study, GEP determined the correct primary site in 9 of the 10 cases (90%), compared to the IHC-guided method which determined the correct primary site for 32 of 50 case evaluations (average 64%, range 50% to 80%). The five pathologists directing the IHC-guided method ordered an average of 8.8 stains per case (range 1 to 18). GEP required an average of 3 slides per case (range 1 to 4).ConclusionsResults of the pilot study suggest that GEP provides correct primary site identification in a higher percentage of metastatic cases than IHC-guided methods, and uses less tissue. A larger comparative effectiveness study using this study design is needed to confirm the results.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1749854104745508

The Who and When of Molecular Testing for Tumors of Unknown Primaries: One Resident’s Perspective

To the Editor It was with great interest that I read the article by Dumur and colleagues1 on their clinical verification of the only US Food and Drug Administration–approved microarray-based test for the diagnosis of poorly and undifferentiated tumors, the Pathwork Tissue of Origin (TOO) Test (Pathwork Diagnostics, Sunnyvale, CA). Briefly, the Pathwork TOO Test is one of several tests currently on the market (CancerTYPE ID, Biotheranostics, San Diego, CA, and miRview mets, Rosetta Genomics, Rehovot, Israel, being others) that attempt to correlate the RNA profile of a given undifferentiated tumor with its suspected tissue of origin (hence, the clever names) through complementary DNA microarray analysis and predetermined algorithms. Based on the findings in the study by Dumur et al,1 the Pathwork TOO test seems to perform glowingly, with a reported 97% concordance rate between the TOO result and final diagnosis, when immunohistochemical and radiologic studies are included. Yet, while most of the available data for these assays are and have been promising with regard to their ability to retrospectively correlate diagnoses with standard analytic methods, several important questions remain, including “When is it appropriate to use one of these tests?” and “Who decides when these tests should be ordered?” As these tests are relatively new to the market and the field of pathology in general, some basic questions are, of course, warranted, especially because they are rather costly, and, as yet, there have been no published prospective studies comparing ultimate outcomes of cases diagnosed with these new techniques vs cases undergoing standard immunohistochemical workups. A recent case seen in our group raises some of these questions and provides a real-life example of when requests for these assays could be made. The patient was a 69-year-old man who had an abdominal computed tomography (CT) scan following a 1-month …

The Pathwork Tissue of Origin TestThe Authors’ Reply

To the Editor We read with interest the article by Dumur et al1 with respect to the use of the Pathwork Diagnostics Tissue of Origin (TOO) Test in the determination of the origin of tumors with unknown primary sites. While this study demonstrates the ability of the test to perform in some clinical settings, we believe that some of the statements and conclusions reached are overreaching and misguided. The authors state that the Pathwork TOO test showed 97% accuracy in determining the site of origin of a tumor, in contrast with immunohistochemical staining, which has a reported accuracy of 75% to 88%,2–4 suggesting that the TOO Test is a better discriminator of the site of origin of tumors. However, in our opinion, this is not a fair comparison. The case types studied are entirely different in these studies. Immunohistochemical panels reached more than 95% accuracy in the correct identification of sites of origin of lung, colon, breast, and prostate carcinoma for which relatively specific and sensitive markers are available. In fact, it would be reasonable to assume that these numbers have only been bettered by the introduction of newer markers, eg, napsin A (lung) and PAX8 (female genital tract). The failure of immunohistochemical staining is limited to relatively specific situations, eg, inability to conclusively identify the site of origin of related tumors such as gastric, pancreatic, and biliary carcinoma for which specific immunohistochemical markers are not available. These “difficult to nail” tumor types were not included in this study, and, as such, the percentages cited are not comparative. In fact, application of an appropriate immunohistochemical panel would have been discriminatory in all 6 cases in which the TOO Test’s performance was cited as being superior. Indeed, the authors concede as much by using immunohistochemical results as …

Estimation of expected survival time using gene expression profiling for tumor site origin.

10584 Background: Expected survival is an important factor in treatment selection and patient decision making. Gene expression profiling for tissue of origin (GEP TOO) can be used to classify metastatic and poorly-differentiated tumors according to the most likely primary site for difficult-to-diagnose tumors. The Pathwork Tissue of Origin Test, (Redwood City, CA) generates Similarity Scores (SS), one for each of the 15 tissues on the Tissue of Origin Test Panel and sum to 100. The highest SS has been validated as the most likely tissue of origin in a large validation study (Pillai et al. 2010). This abstract reports the relationship between the highest SS for a sample and patient survival. Methods: Two cohorts were analyzed in IRB-approved studies. Cohort 1 consisted of 45 patients with carcinoma of unknown primary treated empirically with therapy for carcinoma of unknown primary with known outcome data including survival. Cohort 2 consisted of 107 patients receiving the TOO test in routine clinical practice and included in a decision impact registry study. The relationship between the highest SS generated by the TOO test was examined using parametric (Gamma) and non-parametric (Cox Proportional Hazards) regression models with covariates of age, gender and median survival of the cancer type indicated. Logrank tests were performed to compare survival in patients with SS above or below 50 in each cohort. Results: The highest SS generated by the TOO Test was strongly and positively correlated with patient survival in both Cohort 1 (p = 0.0093, Cox PH) and in Cohort 2 (p=0.0045, Gamma). In Cohort 1, survival for patients with a SS &lt; 50 was 5.9 mos and for patients with a SS ≥ 50 was 9.7 mos (p=0.03, logrank). In Cohort 2, survival for patients with a SS &lt; 50 was 10.6 mos and 22.6 mos for patients with a SS ≥ 50 (p=0.0073, logrank). Conclusions: The Similarity Scores generated by GEP TOO Test have been validated as an aid to diagnosis for difficult-to-diagnose malignancies. Two independent studies of this GEP TOO Test demonstrated a strong positive relationship between the highest SS and survival. This has the potential to expand the clinical utility of this test beyond diagnosis to include prognosis. Further studies to examine this question are underway.

Changes in management and survival outcomes for patients receiving gene-expression-based tissue-of-origin test results for difficult-to-diagnose primary cancers.

397 Background: An important step for a novel test is assessing its clinical utility and real-world effect on diagnosis and patient management. This report describes completed results of a cohort of physicians who have ordered a gene expression profile assay for identification of tumor tissue of origin (Pathwork Tissue of Origin Test) for patients with difficult-to-diagnose primary cancers and their subsequent treatment and survival. Methods: The IRB-approved registry collected data from participating physicians who have ordered the Tissue of Origin test. A detailed interview was conducted using both a web-based questionnaire and a confirmatory telephone interview with the physicians. Chart survey included collection of data on patient demographics, diagnostic procedures including imaging and immunohistochemistry as well as therapy. The physicians were queried regarding their working diagnoses and treatment recommendations prior to and after Tissue of Origin Test result availability. Date of death was also obtained with a minimum of one year follow-up from date of biopsy. Results: One hundred seven patients (61 women) have been studied and 65 participating physicians have completed the interview. Results indicate that most patients underwent extensive evaluation (≥ 10 IHC) prior to gene expression analysis. After receiving test results, physicians changed the primary site diagnosis for 53 patients (50%, CI [43,58%]; p&lt;.001) and changed the treatment management for 72 patients (65%, CI [58%,73%]; p&lt;.0001). At last follow-up, 69 patients had died with a median survival of 426 days. More than 30% of patients were alive at 1000 days. Conclusions: This registry study confirms that in patients with difficult-to-diagnose primary cancers, extensive baseline diagnostics are common and less than half of the patients have an established working diagnosis. Results indicate that the Tissue of Origin Test led to a change in working diagnosis and changed treatment recommendations in over half the patients. Survival outcomes compare favorably to historical controls in treatment trials where median survival is generally less than one year.

Clinical Verification of the Performance of the Pathwork Tissue of Origin Test

Gene expression-based assays have been introduced into the clinical arena to assist in the diagnosis of poorly differentiated or undifferentiated tumors. The US Food and Drug Administration has cleared the microarray-based Pathwork Tissue of Origin (TOO) Test (Pathwork Diagnostics, Sunnyvale, CA) for the molecular characterization of such challenging specimens. We aimed at verifying the analytic and clinical performance of this test on 43 poorly differentiated and undifferentiated tumor samples, including 6 off-panel cases and 7 cancers of unknown primary (CUP). Our results showed 97% (95% confidence interval, 80.4%-99.8%) agreement between the Pathwork TOO Test result and the complete diagnosis, which included clinical correlations and immunohistochemical staining, after the original diagnosis. We concluded that for off-panel and CUP samples, the tissue type and the cell type may be confounded by the Pathwork TOO Test and that careful clinicopathologic assessment is needed when interpreting results from this helpful ancillary tool for pathologists.

Clinical utility of gene-expression profiling for tumor site origin.

e21093 Background: An important step for validating a novel diagnostic is the assessment of its real-world clinical utility. Gene expression profiling (Pathwork Tissue of Origin Test) is recently available to aid in identifying the tumor tissue of origin for metastatic cancers whose primary site is not initially classifiable. The effect of the test on diagnosis and cancer-specific management was assessed in a survey of physicians who ordered the test. Methods: The study was an IRB-approved registry, in which data were collected from participating physicians who ordered the Tissue of Origin Test. To minimize recall bias, a detailed interview was conducted using a web-based questionnaire and a follow-up, confirmatory telephone interview with the enrolled physicians. Data were collected on patient demographics, clinical presentation, diagnostic and imaging procedures, pathology report, immunohistochemistry tests, and cancer-specific management. The physicians were queried regarding their working diagnoses and treatment recommendations prior to and after availability of the Tissue of Origin Test result. Results: Sixty six physicians were enrolled, providing reports on 111 patients (57% women). Patients underwent extensive evaluation, including imaging tests (mean, 3.2) and immunohistochemistry tests (mean, 9.9) prior to gene-expression profiling. The primary diagnosis site changed in 54% (95% CI [46%, 62%]; p<0.0001) of patients after Tissue of Origin testing, and treatment recommendations changed for 65% (95% CI [57%, 72%]; p< 0.0001) of patients. Chemotherapy regimens changed in 61 patients (55%, 95% CI [47%, 63%]; p<0.0001). In 67% of cases, physicians agreed or strongly agreed that the test results were clinically useful. Conclusions: Patients with difficult to diagnose metastatic cancer undergo extensive clinicopathological testing. The addition of Tissue of Origin Test changed a substantial proportion of primary diagnosis sites and treatment recommendations.

Effect of a gene expression-based tissue of origin test's impact on patient management for difficult-to-diagnose primary cancers.

459 Background: An important step for a novel test is assessing its clinical utility and real-world effect on diagnosis and patient management. This report describes the results of a survey of physicians who have ordered a gene expression profile assay for identification of tumor tissue of origin (Pathwork Tissue of Origin Test) for patients with difficult-to-diagnose primary cancers. Methods: The IRB-approved registry collects data from participating physicians who have ordered the Tissue of Origin test for their patients. To minimize recall bias, a detailed interview is conducted using both a web-based questionnaire and a confirmatory telephone interview with the physicians. Chart survey includes collection of data on patient demographics, diagnostic procedures including imaging and pathology including immunohistochemistry as well as therapy. The physicians are queried regarding their working diagnoses and treatment recommendations prior to and after Tissue of Origin Test result availability. Results: Enrollment in the registry is ongoing. Fifty-nine patients (34 women) have been studied and 48 participating physicians have completed the interview. Survey results suggest that most patients have undergone extensive evaluation including multiple imaging tests (median, 6) and exhaustive immunohistochemistry (median, 10) prior to gene expression analysis. For these 59 pts, based on clinicopathologic evaluation, physicians reported working diagnoses for 46% of patients prior to test results and 83% after test results. Treatment recommendations were changed for 53% of patients. The majority (70%) of treatment changes were for chemotherapy. Physicians (n=26) reported that the Tissue of Origin test decreased the diagnosis associated anxiety in 62% of patients. Conclusions: This ongoing registry study confirms that in patients with difficult-to-diagnose primary cancers, extensive baseline diagnostics is not uncommon and less than half of the patients have an established working diagnosis. Survey results suggest that the Tissue of Origin Test led to a change in working diagnosis and changed treatment recommendations in over half the patients. [Table: see text]

Validation and Reproducibility of a Microarray-Based Gene Expression Test for Tumor Identification in Formalin-Fixed, Paraffin-Embedded Specimens

Tumors whose primary site is challenging to diagnose represent a considerable proportion of new cancer cases. We present validation study results for a gene expression-based diagnostic test (the Pathwork Tissue of Origin Test) that aids in determining the tissue of origin using formalin-fixed, paraffin-embedded (FFPE) specimens. Microarray data files were generated for 462 metastatic, poorly differentiated, or undifferentiated FFPE tumor specimens, all of which had a reference diagnosis. The reference diagnoses were masked, and the microarray data files were analyzed using a 2000-gene classification model. The algorithm quantifies the similarity between RNA expression patterns of the study specimens and the 15 tissues on the test panel. Among the 462 specimens, overall agreement with the reference diagnosis was 89% (95% CI, 85% to 91%). In addition to the positive test results (ie, rule-ins), an average of 12 tissues for each specimen could be ruled out with >99% probability. The large size of this study increases confidence in the test results. A multisite reproducibility study showed 89.3% concordance between laboratories. The Tissue of Origin Test makes the benefits of microarray-based gene expression tests for tumor diagnosis available for use with the most common type of histology specimen (ie, FFPE).

Readers' Responses to “Cancer of Unknown Primary Site: Advances in Diagnostics”

agnostic tools used to attempt to identify the primary site of carcinomas, including immunohistochemistry, and acknowledge the limitations of these techniques. 1 They also highlight the recent development of molecular diagnostic tools such as genomic microarray-based Pathwork Tissue of Origin test by Pathwork Diagnostics, using reverse transcription–polymerase chain reaction (RT-PCR)–based assays on paraffin-embedded tissue, which may hopefully enhance diagnostic capabilities, in order to impact on therapeutic decision making. 2 We wish to also highlight the development of microRNA analysis, recently reported at the American Society of Clinical Oncology annual meeting, as an additional tool to aid in molecular diagnosis of cancers of unknown primary site, and propose its availability to the oncologic community at large. Also using RT-PCR, the authors used 22-oligonucleotide RNA molecules (microRNA) involved in gene expression regulation to evaluate RNA extracted from tumors from patients with cancer of unknown primary, and demonstrated concordance (full and plausible) with the actual clinical presentation in 82% of cases. 3 These small RNA molecules have been shown to be highly effective biomarkers, with high tissue specificity, and are more stable for use on formalin-fixed paraffin-embedded block tissue, as well as fine-needle aspiration

Impact of a gene expression-based tissue of origin test on diagnosis and recommendations for first-line chemotherapy.

10579 Background: The Pathwork Tissue of Origin Test for FFPE is a microarray-based gene expression test that aids in the diagnosis of poorly differentiated and metastatic tumors. An accurate diagn...

Identification of tissue of origin in carcinoma of unknown primary with a microarray-based gene expression test

BackgroundCarcinomas of unknown primary (CUP) represent approximately 3%-5% of malignant neoplasms. Identifying the tissue of origin (TOO) in these tumors allows for more specific treatment and improves outcomes. However, primary classification remains a challenge in many cases. We evaluated the ability of a microarray-based gene expression test to identify the TOO in tumor specimens from 21 patients with a diagnosis of CUP.MethodsThe Pathwork® TOO Test was used to measure gene expression patterns for 1550 genes; these were compared for similarity to patterns from 15 known tissue types.ResultsThe TOO Test yielded a clear single positive call for the primary site in 16 of 21 (76%) specimens and was indeterminate in 5 (24%). The positive results were consistent with clinicopathologic suggestions in 10 of the 16 cases (62%). In the remaining six cases the positive results were considered plausible based on clinical information. Positive calls included colorectal (5), breast (4), ovarian (3), lung (2), and pancreas (2). The TOO Test ruled out an average of 11 primary tissues in each CUP specimen.ConclusionThe Pathwork TOO Test reduced diagnostic uncertainty in all CUP cases and could be a valuable addition or alternative to current diagnostic methods for classifying uncertain primary cancers.

Validation of a microarray-based gene expression test for tumors with uncertain origins using formalin-fixed paraffin-embedded (FFPE) specimens

e22015 Background: Microarray-based gene expression has been validated as an aid in the diagnosis of tumors with uncertain origins when the specimen is frozen tissue. Microarray use has been largely limited to RNA derived from frozen specimens. This study evaluated performance of a microarray-based test in identifying the tumor type in FFPE specimens. Methods: ZFFPE human tumor specimens (n=405) representing the 15 tissue of origin sites on the Pathwork® Tissue of Origin Test panel were blinded and evenly distributed between two independent processing labs. All specimens consisted of a 10-μm-paraffin curl containing at least 60% viable tumor and were either metastatic or poorly differentiated primaries. Each specimen was processed through RNA extraction, amplification, labeling, hybridization to a Pathchip® microarray, and was scanned to generate a qualified data file. A pre-specified classification algorithm utilizing more than 1500 genes was applied to each data file to yield Similarity Scores corresponding to the 15 tissues on the test panel. Results were then unblinded and compared to the available diagnoses. Results: Of the 405 specimens, 352 yielded qualified data files (87%). Based on the top Similarity Score, the overall agreement with available diagnoses was 89% (95% CI, 85%-92%) and for each specimen an average of 12 out of 15 tissues could be ruled out with &gt; 99% probability. Results for all tissue types were highly informative with diagnostic odds ratios ranging from 178 to 28509. Performance was similar for metastatic (n=150; 91% agreement) and poorly differentiated primary specimens (n=202; 87% agreement). Conclusions: The large size of this study allows an accurate estimate of the confidence of test predictions for both ruling in and ruling out tissues as likely sites of primary origin. The Pathwork Tissue of Origin Test makes the potential benefits of microarray-based gene expression tests for tumors with uncertain origins available for use with the most common type of histology specimen, FFPE. [Table: see text]

FDA Clears Test that Helps Identify Cancer Type in Tumor Sample

The US Food and Drug Administration (FDA) on July 31, 2008, cleared for marketing a test that can help health care professionals to determine what type of cancer cells are present in a malignant tumor. The Pathwork Tissue of Origin test compares the genetic material of a patient's tumor with genetic information on malignant tumor types stored in a database. The test uses microarray technology to analyze thousands of pieces of genetic material at 1 time and considers 15 common types of malignancy, including bladder, breast, and colorectal tumors. “The clearance of the Pathwork test is another step in the continued integration of molecular-based medicine into standard practice,” said Daniel Schultz, MD, director of the FDA's Center for Devices and Radiological Health, which oversees medical diagnostics. “In the past, scientists have classified different types of cancers based on the organs in which the tumors develop. With the help of microarray technology, they will be able to classify these types of cancers in a standardized non–reader-dependent manner based on the patterns of gene activity in the tumor cells.” The Pathwork Tissue of Origin test is the second in vitro diagnostic multivariate index assay (IVDMIA) device to be cleared by the FDA. In July 2007, the FDA issued a draft guidance document to address premarket pathways and postmarket requirements for IVDMIAs. These tests combine the values of multiple variables to yield a single, patient-specific result. Microarray technology can simultaneously measure gene expression levels of large numbers of genes. Small DNA fragments are placed or arrayed on a slide and then RNA, which has been extracted from the tumor tissue and labeled with a fluorescent marker, is spread over this “microarray.” Because RNA binds to its complementary DNA strand, the level of binding that occurs indicates how active the gene being evaluated is. This is determined by placing the array under a scanning microscope and measuring the intensity of the fluorescent light at each RNA–DNA site on the array. Pathwork's proprietary software converts the scanned image data to gene expression measurements. The gene expression patterns are then compared with known gene expression patterns in the database that correspond to different tumor types. The Pathwork Tissue of Origin test has been demonstrated to provide patterns that confirm the existing tissue of origin of the 15 common tumor types using standard clinical and pathologic information. The accuracy of this test is similar to that achieved by expert pathologists using current standards of practice. PathChip, the gene expression array used in the Pathwork Tissue of Origin test, is custom-designed for Pathwork Diagnostics of Sunnyvale, California, by Affymetrix Inc., of Santa Clara, California. PathChip is the first custom Affymetrix gene expression array to be cleared for diagnostic use.

Interlaboratory Performance of a Microarray-Based Gene Expression Test to Determine Tissue of Origin in Poorly Differentiated and Undifferentiated Cancers

Clinical workup of metastatic malignancies of unknown origin is often arduous and expensive and is reported to be unsuccessful in 30 to 60% of cases. Accurate classification of uncertain primary cancers may improve with microarray-based gene expression testing. We evaluated the analytical performance characteristics of the Pathwork tissue of origin test, which uses expression signals from 1668 probe sets in a gene expression microarray, to quantify the similarity of tumor specimens to 15 known tissues of origin. Sixty archived tissue specimens from poorly and undifferentiated tumors (metastatic and primary) were analyzed at four laboratories representing a wide range of preanalytical conditions (eg, personnel, reagents, instrumentation, and protocols). Cross-laboratory comparisons showed highly reproducible results between laboratories, with correlation coefficients between 0.95 to 0.97 for measurements of similarity scores, and an average 93.8% overall concordance between laboratories in terms of final tissue calls. Bland-Altman plots (mean coefficients of reproducibility of 32.48+/-3.97) and kappa statistics (kappa >0.86) also indicated a high level of agreement between laboratories. We conclude that the Pathwork tissue of origin test is a robust assay that produces consistent results in diverse laboratory conditions reflecting the preanalytical variations found in the everyday clinical practice of molecular diagnostics laboratories.