The current work describes the first validation of a newly developed microRNA-based qRT-PCR test that uses microRNA biomarkers for the identification of tumor tissue of origin. Here, we validated the test in a set of metastatic samples, which were derived from the CNS, of known origin as well as samples from true CUP cases.
Recently, a few papers describing gene expression–based assays to determine the tumor tissue of origin for clinical use were published [22
]. Most studies aimed at identifying tissue of origin used mRNA profiling of tumor samples [26
] and presented the development of the classifier based on a training set and validation results for an independent test set composed of samples that were either not relevant in clinical settings, like primary tumors, or samples that were more clinically relevant, taken from metastatic tissue with known origin. Ma et al. [28
] described the development of a 92-gene qPCR test to differentiate 32 tumor classes that was validated on a small set of 119 independent FFPE tumor samples and resulted in an overall accuracy of 82%. In another study [25
], the tissue of origin was determined based on 1,550 mRNAs measured on microarrays, resulting in an overall sensitivity of 87.8% on a validation set of 547 frozen samples.
Only a few attempts have been made to study the ability of a classifier to determine the tumor tissue of origin in a setting of metastatic tissue derived from real CUP patients. Varadhachary et al. [23
] studied a large cohort of 120 CUP patients using a test that distinguished only six primary sites. The test predicted results for 63 patients, that is, it did not yield a meaningful result in 48% of patients. In another study, Horlings et al. [22
] used an mRNA microarray–based assay in 38 CUP patients and compared the results with tentative suggestions derived from clinicopathological investigation, resulting in consistency with the clinical data for most samples. Here, for the first time, we studied the performance of a molecular test on a consecutive large prospective cohort of metastatic samples from a specific site, the CNS.
In the first phase, we studied test performance in a cohort of samples with “known” tumor of origin. Interestingly, although the test was developed using a training set that contained only a small number of brain metastatic samples, representing only five of the 25 different tumor classes in the assay, the assay performance was very similar to the validation set [21
]. While analyzing the results, we realized that the performance for prostate cancer CNS metastases was much lower than expected. As this manuscript was prepared, a few papers [30
] demonstrated the role of microRNAs in prostate cancer, specifically in antiandrogen therapy and resistance [30
]. Leite et al. [31
] compared the expression of only 14 microRNAs between localized high-grade and metastatic androgen-independent prostate carcinoma and found half of them to be differentially expressed. When we compared microRNA expression, we found that the correlation between the primary and metastatic prostate tissue was significantly lower than seen in other tissues. Because prostate cancer is not considered a common tissue of origin, representing only 2% of CUP cases, as confirmed by autopsy studies [32
], we decided to exclude this origin from further analyses.
Because in CUP, by definition, the primary site in many cases remains unknown, there is inherent difficulty in validating a CUP assay. To tackle this problem, we developed a scoring system that compares the test result with all available clinicopathological data. Based on this scoring system, we demonstrated that, for the vast majority of the true CUP cases, namely, 40 (80%) of 50 cases for which a suggested origin existed, the microRNA-based test was in agreement with the available clinicopathological data.
In some cases, pathological data alone correctly identified the primary tumor site. For such cases, the microRNA test is a confirmative tool. In others, the clinical workup of a patient or the pathology of a metastatic lesion indicated more than one potential primary tumor site, and the microRNA test was helpful in identifying the true primary lesion, as in case ID136. In that case, clinical workup prior to metastatic tumor surgery had detected a lesion suspicious for thyroid tumor. The pathology, however, was in favor of lung cancer as the underlying primary tumor. The microRNA test identified lung as the primary tumor site upfront, resolving the clinical and pathology conflict. Imaging analyses following metastatic surgery identified a lung lesion, histopathologically verified after resection.
Despite extensive workup, including IHC and all currently available clinicodiagnostic procedures, <20% of patients with CUP have a primary site of their cancer identified ante mortem [33
]. Autopsy studies have reported that 70% of cases remain undiagnosed [33
These data impressively show the futility of unguided clinicodiagnostic procedures, which are often time-consuming and expensive. In addition, even though the number of primary tumors likely to metastasize to the brain is limited and a growing number of organ-specific antibodies has helped to elucidate the provenance of metastatic lesions in the majority of cases, 2%–18% of all metastatic lesions to the brain remain tumors of unknown primary site [3
]. This holds true even if taking into account that very practical algorithms for the IHC evaluation of common CNS metastatic neoplasms, including poorly differentiated neoplasms, have been developed. These are common in the way they implement a combination of antibodies to verify or exclude potential primary tumor sites [3
]. These data underscore the necessity of developing diagnostic tests that go beyond IHC.
In a considerable number of cases, the microRNA-based CUP test will be able to do both—identify the potential primary tumor lesion despite ambiguous IHC results and guide clinicodiagnostic procedures—thereby influencing not only diagnostic but also therapeutic decisions in individual patients.
These findings also imply that the primary lesions of CUP tumors that metastasize to the brain are either miniscule at the time of diagnosis and evade diagnostic testing, even at autopsy of the patient, or that CUP tumors may be a distinct group of tumors without true primary lesions but featuring the same metastatic potential as primary tumor lesions outside the CNS. The latter issue has not been extensively investigated on a molecular basis, and the limited information available is still controversial and inconclusive.
There is consent, however, that CUP is a heterogeneous group of tumors that is not a distinct biological entity involving specific genetic and phenotypic alterations. This argues for CUP lesions being derived from miniscule primary tumor sites and argues for the development of more refined molecular tests to identify the true primary tumor location. MicroRNAs are expressed in a highly tissue-specific manner. Because of their small size, microRNAs are robust and stable even in FFPE tissue samples. Thus, microRNAs constitute ideal diagnostic targets for the identification of tissue of origin in otherwise completely dedifferentiated tumors. In our view and experience, investigation of the microRNA profile of a metastatic lesion does not aim to replace either the clinicodiagnostic workup or IHC in surgical pathology or neuropathology but rather significantly extends the diagnostic repertoire of the investigating pathologist to guide clinicians in their search for a primary tumor lesion in true CUP cases lacking organ-specific IHC results. Thus, even when the test results in two possible tissues of origin, it helps the physician to focus the clinicodiagnostic workup to come up with a diagnosis and treatment decision.
Finally, because microRNA profiling is an unbiased analysis investigating a multitude of single independent parameters, it is objective, is investigator independent, and may, in the future, potentially be able to elucidate and define further subsets of metastatic CUP lesions that may follow a better clinical course and may require distinct therapies. Also, with the help of suitable training sets of tumors, the system is open to be extended to tumor types that belong to more favorable subgroups of metastatic CUP lesions that are currently known [37
] and to any favorable subgroup to be identified in the future, to ensure their recognition in clinical practice and allow for specific and individualized treatment and patient management, especially in patients with CUP lesions with a potentially favorable outcome.
As pointed out earlier, a major advantage of the microRNA test is its unbiased nature. In patients who have a history of cancer, the investigating pathologist might be tempted to consider the clinically confirmed primary tumor as the most likely source of the metastatic lesion. In these cases, IHC investigations are aimed at confirming this notion, rather than at identifying other possibilities. This might lead to misinterpretation of the metastases, more so if the primary tumor has already been misinterpreted. Here, a case in the first cohort provides an excellent example. The microRNA test suggested melanoma as the origin, in contrast to the clinicopathological diagnosis of a breast cancer metastasis. This prompted melanoma-specific IHC that was confirmed, not only for the metastatic lesion in the brain but also for the supposedly primary breast tumor.
To date, many anatomic pathology tests, including IHC, have not been extensively studied for their cost-effectiveness [38
]. This lack of information makes an upfront comparison of IHC and any other newly developed diagnostic test in pathology (e.g., the microRNA CUP test presented in this report and mRNA tests) regarding cost-effectiveness difficult, if not impossible. A commonly used matrix to determine the cost-effectiveness of any given diagnostic test is to look at its “cost per increase in patient life expectancy.” If one assumes that all patients with metastatic cancer have a universally poor prognosis and thus a similar life expectancy, then the “cost per increase in patient life expectancy” for any medical procedure in this cohort of patients would likely not be highly cost-effective. However, this assumption is flawed because not all patients with metastatic cancer have a universally negative outcome, nor is life expectancy the most important variable in many cases in which customized palliative treatments based on tumor type would have a profoundly positive impact on quality of life. In addition, increases in patient life expectancy are also likely not the most appropriate measure of an anatomic or molecular pathology test that imparts information rather than a tangible procedure [38
]. Despite all these limitations, it is fair to say that IHC remains, by far, the most inexpensive test in pathology when compared with mRNA- or microRNA-based diagnostic tests. However, this reflects the cost per test only and does not reflect the potential impact of the test on patient management, disease outcome, or patient quality of life. Despite a low cost per test, extensive IHC staining does fail to identify the primary tumor site in metastatic lesions in 3%–10% of cases [36
]. Relative to the costs of other medical tests and procedures in patients with undefined primary malignancies, the microRNA-based CUP test is still rather inexpensive, follows strong and valid biological reasoning, and successfully identifies primary tumor sites in cases with ambiguous IHC results, thereby providing benefits other than and beyond cost-effectiveness, and it may avert even more costly procedures that otherwise would be performed [38
Finally, tumor therapies have become more individualized based on the histomorphological or molecular features of the primary tumor. However, distinct histomorphological subtypes are often clouded by tumor dedifferentiation. Furthermore, clinicians often base their therapeutic strategies on findings in metastatic lesions and refrain from removing additional tissue for tumor classification, even if a primary tumor is identified later. Thus, in practice, the exact classification of the underlying primary tumor rests on the metastatic lesion. Here, refined molecular profiling might prove to be superior to conventional workup in characterizing the actual subtype of the primary tumor. In case ID151, the test succeeded in identifying the true morphological subtype of that particular lung cancer; here, SCLC was validated and confirmed by additional IHC performed following the test results. The small cell nature of the metastatic lesion was missed on conventional pathology.
In conclusion, we demonstrated that this microRNA expression–based assay provides an important objective tool for the diagnosis of tumor tissue of origin in CNS metastases, especially in CUP patients.