An optical photothermal infrared investigation of lymph nodal metastases of oral squamous cell carcinoma

Abstract

In this study, optical photothermal infrared (O-PTIR) spectroscopy combined with machine learning algorithms were used to evaluate 46 tissue cores of surgically resected cervical lymph nodes, some of which harboured oral squamous cell carcinoma nodal metastasis. The ratios obtained between O-PTIR chemical images at 1252 cm^-1 and 1285 cm^-1 were able to reveal morphological details from tissue samples that are comparable to the information achieved by a pathologist's interpretation of optical microscopy of haematoxylin and eosin (H&E) stained samples. Additionally, when used as input data for a hybrid convolutional neural network (CNN) and random forest (RF) analyses, these yielded sensitivities, specificities and precision of 98.6 ± 0.3%, 92 ± 4% and 94 ± 5%, respectively, and an area under receiver operator characteristic (AUC) of 94 ± 2%. Our findings show the potential of O-PTIR technology as a tool to study cancer on tissue samples.

Figure 1

O-PTIR Spectra from points on a single TMA Core. (a) H&E image from an adjacent section for comparison; (b) O-PTIR image showing the averaged signal over four wavenumbers (1252 cm⁻¹, 1285 cm⁻¹, 1540 cm⁻¹, and 1660 cm⁻¹, indicated by vertical lines in (c)); and (c) O-PTIR spectra averaged over the areas of the circles shown in (a) and (b). (i) not heavily keratinised malignant tissue, (ii) lymphoid tissue and (iii) heavily keratinised malignant tissue.

Figure 2

Comparison of resolution of H&E and O-PTIR ratio images. (a) H&E image (pixel size = 0.25 µm); (b) O-PTIR ratio image at 1252 cm⁻¹/1285 cm⁻¹ (pixel size = 1 µm); (c) Higher magnification of the rectangular area in (a); (d) Higher magnification of the rectangular area in (b). Lymphoid tissue appears as mostly dark purple and OSCC appears predominantly as pink. The colours used for the O-PTIR image were chosen to highlight the similarities with the H&E image.

Figure 3

Discriminatory performance of the 1252 cm⁻¹/1285 cm⁻¹ ratio. Three separate lymph node metastases are shown (a–d, e–h and i–l). H&E images (pixel size = 0.25 µm) are shown in (a), (e) and (i); O-PTIR images at 1252 cm⁻¹ (pixel size = 1 µm) are shown in (b), (f) and (j); O-PTIR images at 1285 cm⁻¹ (pixel size = 1 µm) are shown in (c), (g) and (k); ratio of absorbance at 1252 cm⁻¹/1285 cm⁻¹ are shown in (d), (h) and (l). Key to tissue labels: lymphoid tissue (L); stroma (S); OSCC metastasis (T); heavily keratinised area (*); keratin pearl (arrow).

Figure 4

PCA score plot of three annotated classes. (a) Cluster plot of the 46 samples: OSCC metastasis (blue); lymphoid tissue (red); stroma (gold). (b) O-PTIR ratio image of a single core at 1252 cm⁻¹/1285 cm⁻¹. (c) O-PTIR cluster map corresponding to (b) showing tumour (red), cell nuclei (blue) and stroma (gold).