Lateral Flow Assay to Detect Carbonic Anhydrase IX in Seromas of Breast Implant-Associated Anaplastic Large Cell Lymphoma

Abstract

Background/objective: Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) has affected more than 1700 women with textured breast implants. About 80% of patients present with fluid (seroma) around their implant. BIA-ALCL can be cured by surgery alone when confined to the seroma and lining of the peri-implant capsule. To address the need for early detection, we developed a rapid point of care (POC) lateral flow assay (LFA) to identify lymphoma in seromas.

Methods: We compared 28 malignant seromas to 23 benign seromas using both ELISA and LFA. LFA test lines (TL) and control lines (CL) were visualized and measured with imaging software and the TL/CL ratio for each sample was calculated.

Results: By visual exam, the sensitivity for detection of CA9 was 93% and specificity 78%, while the positive predictive value was 84% and negative predictive value 90%. Quantitative image analysis increased the positive predictive value to 96% while the negative predictive value reduced to 79%.

Conclusions: We conclude that CA9 is a sensitive biomarker for detection and screening of patients for BIA-ALCL in patients who present with seromas of unknown etiology. The CA9 LFA can potentially replace ELISA, flow cytometry and other tests requiring specialized equipment, highly trained personnel, larger amounts of fluid and delay in diagnosis of BIA-ALCL.

Keywords: BIA-ALCL; Carbonic Anhydrase IX; lateral flow assay; seroma.

Figure 1

Heatmap comparing concentrations of CA9, CD30 and 12 cytokines in BIA-ALCL and benign seromas.

Figure 2

Dynamic range of CA9 detection using LFA strips. (A) Nine test solutions were prepared by spiking recombinant CA9 into benign seroma samples at defined concentrations, using a 1:2 ratio of seroma to running buffer. Results were imaged using both an iPhone camera (color) and a Bio-Rad imager (black and white) and image analysis was performed using NIH Image J software. (B) The TL/CL ratio exhibited a strong linear correlation with CA9 concentration (Y = 0.13X + 0.45, r² = 0.96, p < 0.0001). CL, control line; TL (CA9), test line.

Figure 3

CA9 Expression in TLBR-1 and TLBR-2 Cell Lines. (A) Immunofluorescence staining of TLBR-1 and TLBR-2 cell lines showed CA9 expression (green), with nuclei counterstained with DAPI in blue. Goat IgG was used as a negative control. (B) LFA analysis demonstrated the presence of CA9 in culture supernatants of both cell lines. CL, control, line; Scale bar = 100 μm.

Figure 4

CA9 LFA analysis on benign and BIA-ALCL seromas. Images were captured using an iPhone camera (color) and the Bio-Rad imager with the Alexa Fluor 488 channel (black and white). Density of CL and TL were determined from Bio-Rad images.

Figure 5

Statistical analysis of CA9 LFA performance. (A) A strong linear correlation was observed between CA9 concentrations measured by ELISA and the TL/CL ratio from the LFA (Y = 0.18X − 0.12, r² = 0.86, p < 0.0001). (B) CA9 levels were significantly elevated in BIA-ALCL samples compared to benign seromas (Benign: 0.38 ± 0.06, n = 23; BIA-ALCL: 0.67 ± 0.19, n = 28; t-test, **** p < 0.0001). (C) Receiver Operating Characteristic (ROC) curve analysis yielded an area under the curve (AUC) of 0.95, indicating excellent diagnostic performance.