Pleural fluid cell-free DNA integrity index to identify cytologically negative malignant pleural effusions including mesotheliomas

Abstract

Background: The diagnosis of malignant pleural effusions (MPE) is often clinically challenging, especially if the cytology is negative for malignancy. DNA integrity index has been reported to be a marker of malignancy. The aim of this study was to evaluate the utility of pleural fluid DNA integrity index in the diagnosis of MPE.

Methods: We studied 75 pleural fluid and matched serum samples from consecutive subjects. Pleural fluid and serum ALU DNA repeats [115bp, 247bp and 247bp/115bp ratio (DNA integrity index)] were assessed by real-time quantitative PCR. Pleural fluid and serum mesothelin levels were quantified using ELISA.

Results: Based on clinico-pathological evaluation, 52 subjects had MPE (including 16 mesotheliomas) and 23 had benign effusions. Pleural fluid DNA integrity index was higher in MPE compared with benign effusions (1.2 vs. 0.8; p<0.001). Cytology had a sensitivity of 55% in diagnosing MPE. If cytology and pleural fluid DNA integrity index were considered together, they exhibited 81% sensitivity and 87% specificity in distinguishing benign and malignant effusions. In cytology-negative pleural effusions (35 MPE and 28 benign effusions), elevated pleural fluid DNA integrity index had an 81% positive predictive value in detecting MPEs. In the detection of mesothelioma, at a specificity of 90%, pleural fluid DNA integrity index had similar sensitivity to pleural fluid and serum mesothelin (75% each respectively).

Conclusion: Pleural fluid DNA integrity index is a promising diagnostic biomarker for identification of MPEs, including mesothelioma. This biomarker may be particularly useful in cases of MPE where pleural aspirate cytology is negative, and could guide the decision to undertake more invasive definitive testing. A prospective validation study is being undertaken to validate our findings and test the clinical utility of this biomarker for altering clinical practice.

Figure 1

Standard curves of ALU 115bp and 247bp amplicons. DNA from female genomic DNA was serially diluted (from 10ng to 0.1ng) and used to construct the standard curves from which the quantity of DNA of the patients was calculated.

Figure 2

Melting curves of ALU 115bp and ALU 247bp amplifications signals from 20 patients. Following amplification, melting curves were obtained by calculating the derivatives dF/dT vs. temperature. ALU 115bp primers produce a peak of approximately 83°C and ALU 247bp at approximately 86°C.

Figure 3

Box plots of pleural effusion DNA fragments, integrity index and mesothelin concentrations in subjects with effusions due to benign aetiology, other cancers and mesothelioma. DNA fragment values were determined by quantitative PCR for ALU (a) 115bp fragment and (b) a 247bp fragment. The DNA integrity index (c) was defined as the ratio of ALU 247/ALU 115 fragment levels. Mesothelin values (d) were determined by ELISA. Mann-Whitney U test to was used to assess for any differences in comparison to benign effusions and p values <0.05 were deemed significant.

Figure 4

Box plots of serum DNA fragments, integrity index and mesothelin concentrations in subjects with effusions due to benign aetiology, other cancers and mesothelioma. DNA fragment values were determined by quantitative PCR for ALU (a) 115bp fragment and (b) a 247bp fragment. The DNA integrity index (c) was defined as the ratio of ALU 247/ALU 115 fragment levels. Mesothelin values (d) were determined by ELISA. Mann-Whitney U test to was used to assess for any differences in comparison to benign effusions and p values <0.05 were deemed significant.

Figure 5

Dot plot representing the correlation of mesothelin (log values) concentration of pleural effusion and serum in study subjects (n=74).

Figure 6

Receiver operator characteristic analysis of pleural effusion DNA integrity index, pleural effusion mesothelin and serum mesothelin to evaluate the diagnostic accuracy of malignant pleural effusions (a) and mesothelioma (b).