FNA biopsy, whether image-guided or performed by direct palpation, is a clinically common and useful diagnostic method in patients with presumed neoplasms. It is less invasive than an open biopsy or a core needle biopsy and is, therefore, associated with less discomfort and a lower risk of bleeding, pneumothorax, or viscous perforation (17
). However, the accuracy, specificity and sensitivity of the cytological interpretation of FNA biopsies may be limited in a variety of circumstances such as target size, tumor type, accessibility etc. One major limitation is the need for intact recognizable tumor cells and architecture for definitive histological typing and diagnosis (12
). Even when tumor is detected, the histological subtype and features associated with prognosis may not be ascertainable. We hypothesized that the addition of molecular tests such as gene expression analysis to the cytological analysis for FNA biopsies would potentially enhance the sensitivity and specificity of this method for cancer diagnosis as well as for application of prognostic and pharmacogenomic tests.
We previously reported that the combination of a small number of carefully chosen and validated gene expression ratios can be used to develop diagnostic and prognostic tests for cancer. We proposed specific tests for diagnosis of MPM, lung cancer, bladder cancer and prostate cancer as well as prognostic tests for MPM, lung cancer and breast cancer (8
). The prognostic and diagnostic tests for MPM were recently validated in a prospective clinical trial using tissue biopsies demonstrating that when at least 3 biopsies are used per patient, these tests are highly accurate (9
). The prognostic test was independent in the multivariable model from lymph node status, stage and histological subtype. Since these molecular tests require the simultaneous measurement of expression of only a few genes, and it is the ratio of the gene expression levels that is the key variable, we hypothesized that this type of test could be expeditiously measured in FNA biopsy specimens. We previously demonstrated the feasibility of this approach in distinguishing lung cancer from normal adjacent lung using a small number of ex-vivo
FNA biopsies (21
). Herein, we examined and tested this approach in MPM tumors where traditional FNA based diagnosis has been inadequate. First, we determined how much RNA was required for reproducible results utilizing RNA extracted from cell lines. Then, we investigated whether a gene expression ratio test was capable of differentiating MPM from lung cancer using ex-vivo
FNA biopsies. Finally, we examined for the first time the accuracy of a gene expression ratio-based prognostic test for MPM using FNA biopsies.
In this report, we demonstrate that the FNA biopsy technique captures sufficient RNA for RT-PCR analysis in the majority of cases but that there is considerable variability in the RNA amount extracted per sample, which is more likely linked to the technical performance of the procedure rather than to the specific tumor evaluated. We established that, at least for the ratio tests examined, 500 ng of total RNA provides sufficient starting material for reproducible results and that a MPM diagnostic test applied to a large number of ex-vivo
FNA biopsies from MPM and adenocarcinoma is 100% sensitive and 90% specific. We also examined the performance of a prognostic test that had previously been prospectively validated for MPM in solid tumor biopsies (9
). We demonstrated that the test was feasible using 500 ng of starting total RNA and that it was concordant in the majority of the cases with the same test applied to matched solid tumor specimens. While the FNA-based classification was 74% concordant with surgical specimens overall, the specificity for good risk patients was particularly high at 97%. MPM is a highly lethal tumor for which few therapies are effective (5
), therefore, it is important to identify upfront those patients who might benefit from aggressive surgery. Thus, the finding that the FNA approach is quite effective in identifying the good risk patients is associated with high clinical utility.
There are several limitations of this study. One is that the biopsies were done in ex-vivo specimens and it is possible that the RNA yield or tumor accessibility of in-vivo FNA speciemens will be less adequate. However, the success of the ex-vivo biopsies suggests that with careful modulation of the image guidance technique and the biopsy procedure this approach can be translated into an image-guided in-vivo FNA biopsy procedure. This hypothesis is currently being addressed in a clinical trial at our institution. Another limitation is that while correctly identifying the good prognosis patients, the prognostic test was insufficiently specific for the poor prognosis patients. This test property can be potentially overcome by either combining the molecular test information with additional radiological and clinical tests to design risk stratification algorithms and/or by performing new discovery experiments to define better molecular tests specific to FNA biopsies.
Several reports have described the application of direct or ultrasound-guided FNA combined with molecular testing in breast (22
) and thyroid cancer (23
). A pilot study has been conducted using FNAs from primary breast cancers to identify quality control criteria for individual microarray and to assess whether gene expression profiles obtained from the FNAs were representative of the tumors (24
). Although the results displayed that only 15% of the FNAs provided enough mRNA for the expression array analysis, the study established some guidelines to combine FNA with cDNA microarray technology. In addition, the amplification of the extracted RNA was successfully utilized in another FNA study to examine pathways of chemoresistance in breast cancer but not for diagnosis or prognosis (25
One of the most commonly used FNA-based cytological tests is for the diagnosis of thyroid cancer within thyroid nodules. Unfortunately, up to 30% of thyroid FNA biopsies are indeterminate and these patients often require a diagnostic thyroidectomy (26
). A recent study has been performed to determine the diagnostic accuracy of a 3-gene assay in thyroid tumor tissue and in clinical FNA biopsy samples for distinguishing benign from malignant thyroid neoplasms (27
). Although 2 of the 3 proposed gene markers were significantly differentially expressed in malignant thyroid tissue and clinical FNA biopsies, the characteristic curve analysis for these 3 genes, individually or in combination, did not display sufficient accuracy to distinguish benign from malignant thyroid neoplasms in either FNA biopsy or tissue samples. Another effort to discriminate between benign follicular thyroid adenoma and malignant follicular thyroid carcinoma by using FNA biopsy analysis at the transcript level has been reported by Cerutti et al
). Although the main focus was on the immunohistochemistry of four proteins, the transcript levels of the corresponding four genes were analyzed by RT-PCR. The results showed that even if the transcript levels reliably predicted malignancy, the immunohistochemical analysis had higher sensitivity.
FNA biopsies are an ideal method for detecting tumor and following patients during the life-cycle of the cancer. They are minimally invasive, can be performed under local anesthesia and minimal sedation, and on an outpatient basis whether image-guided or not. Theoretically, they can be used for detection, diagnosis, determining prognosis, therapy and cause for failure. FNAs can be obtained directly when a lesion is palpable or under many types of image-guidance (12
). In the thoracic cavity, image-guidance techniques may include conventional ultrasound, computerized tomography (CT), MRI and endoscopic guidance as by endo-esophageal and endo-bronchial ultrasound. For many decades cytological analysis has been successfully applied to FNA biopsies with some limitations.
In the present study, we demonstrate for the first time that sufficient RNA can be extracted from most FNA biopsies to allow for gene-ratio based analysis using specific diagnostic and prognostic tests in MPM and lung cancer. We envision this method as an adjunct to increase the accuracy and spectrum of tests that may be done via this approach for thoracic cancer specifically and all cancers in general.
Statement of Translational Relevance
Cytological evaluation of specimens obtained by fine-needle aspiration (FNA) is a useful method to diagnose malignancies in patients with accessible lesions. To improve the accuracy of FNA-based diagnosis, previous investigations have attempted to combine FNA biopsies and molecular techniques with limited success. In this report, we describe a large study designed to determine the accuracy of diagnostic and prognostic gene expression ratio tests in malignant pleural mesothelioma (MPM) using RNA from ex-vivo FNA biopsies. Despite considerable variability in the amount of extracted RNA among FNA samples, the ratio-based algorithms were capable of differentiating MPM from lung cancer and predicting the outcome of MPM patients. We conclude that the minimally invasive FNA procedure may be coupled with molecular-based tests to improve diagnosis, allow for prognostic tests, and ultimately aid in the management of cancer patients.