Background
Metastatic tumors to the brain are the most common central nervous system (CNS) neoplasm and occur in about 15% of all cancer patients. In an adult, these tumors originate most frequently from lung, breast, skin, kidney and colon [
1]. In up to 10% of patients, no clear primary site can be determined despite an intensive clinical evaluation [
2,
3]. Current treatment regimens for these patients with unknown primaries include surgery, radiation and chemotherapy [
4,
5]. Despite these treatments, the survival rates of patients with brain metastases remain low. A recent retrospective review of 740 patients with brain metastases of all tissue types reported actuarial survival rates of 8.1% at 2 years and 2.4% at 5 years [
6].
The last decade has seen the emergence of a number of markers of immunohistochemistry (IHC) that have been helpful in indicating the origin of common metastatic tumors to the brain [
7,
8]. Workup of an immunohistochemical signature requires selection and use of a large number of antibodies and knowledge of their reactivities (and cross-reactivities) to different cancer types. A recently proposed algorithm for immunohistochemical evaluation of the common types of brain metastases recommends the use of 18 antibodies to distinguish 10 cancer types [
7]. Immunohistochemical markers with a high degree of sensitivity and that are also highly specific for a single primary site are uncommon. In addition, interpretation of immunohistochemical stains is user-dependent and requires subjective interpretation. Even after a rigorous workup, success rates at identifying primary tumor sites are not optimal [
9]. A meta-analysis of four studies in which pathologists were blinded to the knowledge of the primary site showed that even an extensive IHC workup correctly identified the primary site for only 66% of all metastatic specimens [
9]. Additional diagnostic approaches are required to complement more traditional IHC analysis.
Diagnostic molecular profiling assays that use either microarrays or real-time reverse transcription polymerase chain reaction (RT-PCR) have been developed to identify the tissue of origin of metastatic cancers [
10,
11]. Among these, microarray-based assays have the advantage of simultaneously evaluating the gene expression pattern of thousands of genes. The information generated is mined using multigene classifiers that predict the tissue of origin. The Pathwork
® Tissue of Origin Test (Pathwork Diagnostics, Redwood City, CA, USA) for frozen specimens uses microarray technology to measure the gene expression pattern, comprising 1550 genes, of a tumor with an uncertain origin and compares it to expression patterns of a panel of 15 known tumor types. The tissue types represented are: bladder, breast, colorectal, gastric, hepatocellular, kidney, non-small cell lung, ovarian, pancreatic, prostate, thyroid, melanoma, testicular germ cell, non-Hodgkin's lymphoma, and sarcoma. This panel represents approximately 90 percent of all solid tumors and 58 morphologies overall. In validation studies, the Tissue of Origin Test had an accuracy of 87.8% in a set of 547 frozen specimens and delivered reproducible results (93.8% concordance) in four different laboratory settings [
10,
12].
The purpose of this study was to evaluate the performance of the Pathwork Tissue of Origin Test in identifying the primary site for metastatic brain cancer patients. Fifteen cases of metastatic brain cancers of known origins were processed through the Tissue of Origin Test, and predicted the accurate tissue of origin in 92.3% of cases that met specimen entry criteria. These results show that the Tissue of Origin Test is a useful diagnostic tool to aid physicians treating metastatic brain cancer patients.
Methods
Tumor Specimens
Tumor specimens that met the following criteria were entered into the study: excisional biopsies of brain metastases, fresh-frozen (100 mg) samples available for analysis, sample contained at least 60% tumor and < 20% necrosis and the primary site of the tumor was known. Sixteen fresh-frozen brain metastases were obtained from the UCSF Neurological Surgery Tissue Bank using protocols approved by the UCSF Committee of Human Research. One additional frozen brain metastasis tumor specimen was obtained from Cytomyx (Rockville, MD). In addition, microarray data files for seven frozen brain metastases were obtained from Gene Logic, Inc. (Gaithersburg, MD) and one from Gene Expression Omnibus (GEO; Accession number GSM76622). When available, Hematoxylin and Eosin (H&E) sections adjacent to the tumor sample were reviewed by a pathologist to determine the percentage of tumor cells and necrosis. In all cases, the primary tissue site of the brain metastasis was known.
Specimens were processed as described earlier [
10,
12]. Total RNA was extracted using the RNeasy Midi kits (Qiagen Inc., Valencia, CA) following manufacturer-recommended protocols and as described earlier [
12]. Total RNA concentration was assessed by spectrophotometry (OD 260 nm), and the purity was judged by the ratio of absorbance at 260 nm to 280 nm (A
260/A
280). Biotin-labeled cRNA was synthesized using GeneChip expression assay reagents and protocols (Affymetrix, Inc, Santa Clara, CA), and the samples were hybridized to the Pathwork Diagnostics Pathchip using commercially available reagent kits and protocols (Affymetrix, Inc., Santa Clara, CA). The arrays were scanned using the Affymetrix GCS3000 Scanner. The resulting raw intensity data files (CEL files) were processed at Pathwork Diagnostics for automated analysis and report generation. The CEL files contain raw data on RNA expression in each tumor. Only CEL file that have greater than a minimum threshold of overall signal of 20 are used for further analysis. The Tissue of Origin Test utilizes the expression levels of 1550 genes to compute a similarity score to each of 15 cancer types as described earlier [
10].
Discussion
The Pathwork Tissue of Origin Test showed a high degree of agreement with the clinically available diagnosis, accurately identifying the primary site for 92.3% of the metastases to brain from primary sites on the Tissue of Origin panel and 86.7% overall (including the off-panel head and neck carcinoma). This rate of agreement is quite comparable to the percent agreement of 87.8% obtained during the clinical validation of the Tissue of Origin Test [
10] and is superior to the 66% agreement seen in similar blinded studies in which a pathologist uses immunohistochemistry to identify the primary [
9]. In addition to providing information about the most likely primary site, each Test also provided information that definitively ruled out an average of 13 tissues as sites of origin.
The cases in this study were not selected for having primary sites on the Tissue of Origin Test panel. Nonetheless, 93% of the specimens' primary sites were available on the Tissue of Origin Test panel. For the off-panel specimen, the Test generated 13 tissue rule-outs. The Tissue of Origin Test results were also consistent with the expected distribution of primary sites for the tumors most commonly metastasizing to brain [
1].
Currently, a version of the test has become available that works with formalin-fixed paraffin-embedded (FFPE) specimens [
13]. The ability to use FFPE enhances the clinical utility of the Tissue of Origin Test since FFPE is the more commonly available clinical material. While the method of processing the total RNA and classification algorithm used are different in the frozen and FFPE versions of the Tissue of Origin Test, they both use microarray expression data and multigene classifiers to distinguish between the same 15 tissue types described in this study. The FFPE Test output, similar to the frozen Test, is 15 similarity scores that range from 0 (very low similarity) to 100 (very high similarity). Importantly, clinical validation studies have shown that performance of the FFPE Test had an accuracy of 88.5% in a set of 462 FFPE specimens which is equivalent to the frozen Test [
10,
13].
A large number of screened samples were not eligible for entry into the study because they contained extensive necrosis. The validation studies for the Tissue of Origin Test did not include samples with necrosis > 20%. Tumor necrosis in brain metastases is often extensive, leaving recognizable tumor tissue only at the periphery of the lesion and around blood vessels. Tissue macrodissection to remove necrotic tissue performed by a trained pathologist might be used successfully with the Tissue of Origin Test for highly necrotic samples. The FFPE version of the Tissue of Origin Test can be used with specimens containing up to 40% necrosis (minimum 60% tumor tissue) making it more useful in clinical practice for brain tumors. Additionally, tumor punches of FFPE tissue blocks can be performed to select non-necrotic tumor tissue for the Tissue of Origin Test.
Our study shows that the Tissue of Origin Test is a useful diagnostic tool for brain metastases and can serve as a tool for neuropathologists as they classify metastatic brain cancers. It is expected that improved accuracy in the diagnosis of the primary sites for metastatic brain cancer will aid in selecting further diagnostic tests and optimal therapy. Not only would it guide the clinical workup to locate the primary tumor but it would allow oncologists to treat these patients with more specific targeted therapies. Several cancers that commonly metastasize to the brain such as breast, ovarian and small cell lung carcinomas are highly responsive to current therapies and an accurate diagnosis would be of considerable benefit to these patients. Accurate identification of the primary site could eventually lead to improved clinical outcomes. It has been shown that identifying colon as the primary site for carcinoma of unknown primary (CUP) patients results in improved clinical outcome [
14,
15]. These CUP patients had better response to colon-cancer specific treatment regimens than they did to empiric CUP therapy with paclitaxel and carboplatin-based regimens.
Competing interests
Three of the authors are employees of Pathwork Diagnostics, manufacturer of the Tissue of Origin Test.
Authors' contributions
AHBW, WDH, and RP conceived and designed the study, and reviewed the manuscript. JCD assisted in the performance of the study, interpreting the data, and reviewed the manuscript. HW assisted in the performance of the study. SRV was the custodian of the UCSF Neurological Surgery Tissue Bank, reviewed the pathology of the samples, and provided clinical expertise on these cases. AL participated in interpreting the data and authored the manuscript. All authors have read and approved of the final manuscript.