This initial study demonstrated that the use of commercially available MPM enabled detailed imaging of the PCs at the tumour-parenchymal interface of RCCs to facilitate precise measurements of its thickness and collagen characteristics. At the tumour-parenchymal interface, there was significant variation in the thickness and hence median collagen area of the PC between the thinnest and thickest regions of interest within each tumour. Clear cell tumours were found to have significantly median collagen fibre densities than non-clear cell tumours at both the thinnest and thickest regions of interest in the PC.
Multi-Photon Microscopy is a highly accurate tool for imaging of collagen and analysing its organization from molecular scale up to tissue architectural level. It combines the advantages of a non-linear imaging approach with a coherent modality able to probe molecular organization.12–13 Another key advantage is the ability to process fresh, unstained tissues without the need for staining.5 Historically, pathologists have focused on the morphological characteristics of cancer cells under light microscopy, such as their architectural growth patterns and cytological features, in the subtyping and grading of tumours. However, with the high sensitivity and specificity in probing collagen characteristics, MPM has increasingly been employed in various cancer tissues. It has been suggested that MPM has the ability to grade, differentiate and prognosticate certain cancers, for example breast,14 ovarian15 and gastric cancers.16
Multi-Photon Microscopy has also been employed in urological cancers. Ling et al found that Gleason grade in Prostate cancer could be correlated with the ratio of anisotropic and isotropic collagen fibres in biopsy tissues imaged with MPM, potentially serving as a biomarker for cancer aggressiveness.17 Brooks et al used MPM to investigate collagen structure of non-muscle invasive bladder cancer, demonstrating different collagen fibre morphology in patients with subsequent invasive progression, potentially serving as a prognostication and treatment decision tool.18
MPM has been previously used to image RCC tumour specimens, without analysis of the pseudocapsule. Best et al demonstrated higher collagen fibre density and alignment in grade 4 compared to grade 1 RCC, and suggested that MPM could complement traditional tumour grading in future.19 In a study by Jain et al, MPM demonstrated ability to differentiate tumor from normal parenchyma in fresh, unstained kidney tissues. In their study, it distinguished RCC histological subtypes based on their tissue architecture, with a diagnostic accuracy of 95%.20 It was suggested that MPM may potentially be used for real time unstained kidney tissue biopsy diagnosis and surgical margin assessment in future.20
The findings in our report support the other reports that the presence and integrity of RCC pseudocapsules are known to vary between tumours. Higher rates of PC presence and PC completeness have been found in clear cell RCC.3 Higher risk of PC invasion have been demonstrated in larger tumors and tumors with incomplete PC.21 PC invasion has also been associated with higher grade and stage of tumors, which independently predict the risk of tumor recurrence.1 Kim et al studied 258 patients with clinical T1a tumours who underwent standard partial nephrectomy. It was found that 26.7% of tumours had incomplete PCs, 14.3% had absent PCs, and 17.4% had complete PC invasion.22 However, the reason why certain tumour subtypes and higher grade tumours have incomplete PCs or have higher rates of PC invasion remain poorly understood. Our studies suggest that the PCs in RCCs with clear cell histology may be tougher with significantly greater collagen fibre density. This suggest that RCCs with clear cell histology may have tougher PCs than non-clear cell histology.
Indeed, there are few studies analysing the characteristics of PCs beyond just structural integrity. Provenazano et al described the process of increased extra-cellular matrix collagen deposition, remodelling and crosslinking to resist tumour expansion.23 Wang et al studied of PCs in small renal masses, and established collagen and reticulin to be abundant within the fibrous stroma of PCs. Pseudocapsules were shown to be a rim of compressed fibrous tissue at the tumour-parenchymal margin. Through special staining with Tri-chrome, they demonstrated consistently high collagen content in PCs across all tumour histological subtypes.24 The high collagen content is the reason why MPM is potentially a game-changing tool for providing insights into the characteristics and biological behaviour of PCs. For example, in addition to inter tumour variations, our study showed that the RCC PC has significant thickness and collagen area variation even within each tumour along the tumour-parenchymal interface. Despite the differences in PC thickness, collagen fibre density and the complexity of linkages between the fibres (as measured by the reticulation index) are not significantly different between the thinnest and thickest areas of PC for each tumour. Intriguingly, this suggest that the site of any PC invasion by its tumour is likely a function of the thickness alone and not ‘structural’ weakness.
This is the first study to employ the stain-free MPM technology to analyse collagen characteristics of RCC pseudocapsules. Our data suggests that each RCC PC is not a consistent layer when fine collagen characteristics are examined using the stain-free MPM technology. Even with a truly complete and intact PC, we have shown that there are quantifiable and demonstrable intra-tumoral variation of collagen quantity and quality within each tumour PC. This has implications on the technique of Tumour Enucleation, which relies on the complete dissection along the fibrous PC. The PC, being the boundary between normal tissue and tumour cells, is a key factor during enucleation and surgeons should be aware of such intra-tumoral variation in PC thickness to achieve precise and complete tumour enucleation. Despite favourable published data, concerns over the oncological safety and positive margin rates of enucleation remain, mainly related to the integrity of the PC.1,22 Indeed, intertumoral differences of collagen density between clear and non-clear cell cancers in our study suggest different pseudocapsular toughness when exposed to surgical dissection during enucleation. This may be the reason why anecdotally, surgeons may have avoided enucleation for papillary cell cancers for fear of rupturing the weaker PCs, resulting in tumour spillage.
The main drawback of the study is the small numbers and the retrospective nature. A larger number over a longer study period would enable us to better correlate collagen characteristics with different tumour histological subtypes, grades and stages. Indeed, with increasing use of better imaging techniques, it is anticipated others will utilize MPM to corroborate and extend our initial findings.