Accurate classification of metastatic brain tumors using a novel microRNA-based test

Abstract

Background: Identification of the tissue of origin of a brain metastatic tumor is vital to its management. Carcinoma of unknown primary (CUP) is common in oncology, representing 3%-5% of all invasive malignancies. We aimed to validate a recently developed microRNA-based quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) test for identifying the tumor tissue of origin, first in a consecutive cohort of metastatic tumors of known origin and then in a cohort of CUP cases resected from the central nervous system (CNS).

Patients and methods: One hundred two resected CNS metastatic tumors with known origin, previously classified based on the patient's clinical history and pathological data, as well as a second cohort of resected CNS tumors from 57 patients originally diagnosed as CUP were studied. A qRT-PCR diagnostic assay that measures the expression level of 48 microRNAs was used to classify the tissue of origin of these metastatic tumors.

Results: In this blinded study, the test predictions correctly identified the reference diagnosis of the samples of known origin, excluding samples from prostate origin, in 84% of cases. In the second CUP patient cohort, the test prediction was in agreement with the diagnosis that was later confirmed clinically or with pathological evaluation in 80% of cases.

Conclusion: In a cohort of brain and spinal metastases, a previously developed test based on the expression of 48 microRNAs allowed accurate identification of the tumor tissue of origin in the majority of cases. The high accuracy of this test in identifying the tissue of origin of metastases of unknown primary is demonstrated for the first time and may have broad clinical application.

Figure 1.

Confusion matrix for the 52 phase 1 samples resulting in a single answer. Rows indicate the reference diagnosis tissue and columns indicate the tissue determined by the microRNA test (shown are only the origins relevant for the single-answer cases). Numbers in parentheses (along the y-axis) denote the overall sensitivity per tissue of origin among the single-answer cases.

Figure 2.

MicroRNA median C_T signals in central nervous system metastases from phase 1 compared with primaries from the set used to develop the assay. Signals are normalized C_T values. Dotted lines indicate factor 2. Analysis excluded three brain-specific microRNAs (miR-124, miR-9*, and miR-138), because we assume that their expression was derived from the surrounding tissue. Abbreviation: C_T, cycle threshold.

Figure 3.

Classification example. (A): Measured levels (normalized C_T, inversely proportional to log(abundance)) of hsa-miR-200c and hsa-miR-148b are compared for all training set samples and the tested sample. (B): Hematoxylin and eosin staining (upper panel) showed that the metastasis was composed of undifferentiated pleomorphic tumor cells. These reveal focally strong and specific immunopositivity for S100 (lower panel) and HMB45 (middle panel). Abbreviation: C_T, cycle threshold.