We present here an improved assay for prediction of the tissue of origin in metastatic samples. The second-generation assay employs the expression of 64 microRNAs to predict 42 tumor types, covering >92% of all solid tumors [27
]. The assay uses a custom-designed microarray and the results were highly reproducible when the assay was performed in two laboratories. The overall accuracy of the assay, based on an independent validation set of 509 samples, was high (85%), with 82% of the samples producing a single predicted origin with 90% accuracy. The assay was also validated on a set of CNS metastatic samples of patients with CUP, resulting in 88% concordance with the clinicopathological evaluation of the patients—an extremely high concordance compared with published studies looking at different genomic profiling approaches for diagnosing the tumor of origin in patients with CUP [9
Our previous experience developing a clinical assay for the identification of the origin of metastatic tumors, which identifies the tissue of origin from 17 organs with a total of 25 histologic subtypes [22
], has shown proven usefulness in clinical studies that demonstrate the high accuracy of the molecular profiling results [23
]. Even though the most common primary tissues of origin for CUP were represented in our first-generation assay, there was a desire to improve its clinical utility by including other carcinomas, such as urothelial carcinoma, carcinoma of the uterine cervix, additional histological subtypes for renal cell carcinoma, adrenocortical carcinoma and pheochromocytoma, and different types of sarcoma, mesothelioma, lymphoma, and primitive germ cell tumors of the ovary.
One of the challenges for the development of FFPE-based assays with retrospective samples is that older archival blocks may not provide RNA of sufficient quality to obtain meaningful results. Penland et al. reported successful mRNA expression analysis using microarrays in only one-quarter of unselected FFPE blocks that were between 2 and 8 years old [30
]. More recently, much higher failure rates have been described for mRNA-based expression used for clinical commercial assays: 22% for Pathwork CUP assay [10
] and 6%–29% for BioTheranostics CUP assay [28
]. The QA failure rate for the microRNA-based assay presented here is 2% (11 of 509 cases of known origin and 1 of 55 patients with CUP) for specimens 1–20 years old without reduction in the quality of the RNA extraction or the accuracy of the assay results (supplemental online Fig. S4).
Potential issues were recently raised [28
] about the use of microarray platform as compared to qRT-PCR, claiming lower sensitivity, batch effects, and a limited dynamic range of 102
. These limitations were all indeed demonstrated for mRNA microarray measurement. In contrast, our microRNA microarray platform (supplemental online Fig. S1) demonstrated an extremely high reproducibility (at least 10 different batches of microarrays were used), sensitivity, specificity, and a dynamic range of >103
, thereby demonstrating the validity of this platform for use in a clinical setting.
Another potential issue often raised regarding the development and validation of molecular profiling assays is the number of specimens used. The miRview mets2
assay presented here was developed based on 1,282 tumor samples and validated on a cohort of 509 tissue specimens that was independent of the discovery and training cohort. The size of the validation cohort is similar to the 547 tissue samples used for the validation of the Pathwork CUP [10
] assay that uses an Affymetrix microarray platform [32
] and significantly more than the 187 samples used for the validation of the bioTheranostics CUP assay that uses a qRT-PCR platform [28
]. The number of specimens per tumor class that were used in the training phase of the assay ranged from 5 to 140 (median 24); in the validation cohort, the range was 2–26 samples per tumor class (median 15; ). The tumors with the smallest numbers were typically subgroups of larger categories (e.g., different types of sarcomas). The validation panel included primaries and metastases from different differentiation levels, including poorly and undifferentiated tumors.
The assay was further validated on a cohort of actual CUP patients, previously studied on the first version of the microRNA-based assay [23
]. This validation confirms the high level of accuracy of microRNA-based profiling in CUP cases that we have seen in the earlier study and also demonstrates the improvement of the new assay with an overall concordance to the clinicopathological evaluation in 88% of the samples compared with 80% concordance in the previous study. This high level of concordance can be compared to other commercial tests, which have similar performance in validations based on known primaries but show marked deterioration in performance when testing real patients with CUP. Pathwork reported 62% concordance [33
] and bioTheranostics reported 75%–76% concordance [28
], compared with the 88% concordance when using the miRview mets2
Molecular profiling in CUP should be considered in the context of IHC, which is a standard diagnostic method used to determine tissue origin. IHC is a powerful tool in CUP [32
] cases, but even with the use of IHC, there remains a need for additional diagnostic methods. The choice of the IHC panel itself is a subjective decision that may be biased by the clinical history and presentation of the patient. Interpretation of the IHC results is also subjective, resulting in high interobserver and intraobserver variability. The objective and unbiased approach of this assay is a major advantage, as well as its high reproducibility demonstrated in the interlaboratory results comparison. Moreover, in >30% of the cases, the staining pattern of IHC does not result in a conclusive diagnosis [12
]. This may be the case for tumor locations for which no specific markers are available or dedifferentiated tumors which have lost expression of characteristic markers. The fact that we found no deterioration of performance of our assay between cases of known primary and CUP cases that are more difficult to diagnose suggests that this molecular assay adds information to that obtained by IHC. Thus, the miRview mets2
assay may complement IHC and guide diagnosis in difficult or uncertain cases, especially when IHC studies are inconclusive or incompatible with clinical findings.
Finally, any given assay able to predict tissue of origin with high sensitivity and specificity is potentially interesting for clinical oncologists. It is the more practical issues, however, that determine its definite clinical implementation in day-to-day practice. One major issue with expression platform-based analyses is time. Ideally, the timeframe from obtaining the tissue to the decision to process the tissue on the platform to the result of the platform analysis guiding all further clinical decisions should not exceed the time usually needed for a standard pathology workup of a surgically obtained specimen. The total turnaround time for the miRview mets2 assay is 7–10 days, which is a timeframe well suited to meet clinical needs. In addition, in the case of patients with cancers of unknown or uncertain primary origin, this short processing time allows unguided tumor evaluation and staging investigations to be put on hold until the analysis data are available. Besides better guiding patient management and therapy, this might also help reduce constantly growing evaluation costs in patients with cancers of unknown or uncertain primary origin.
In summary, this improved second-generation microRNA-based assay can serve as a reliable diagnostic tool to aid physicians with challenging diagnostic dilemmas.