Remodeling of the ECM is a central mechanism in the progression of IPF, and remodeling of type VI collagen has been suggested to be associated with disease progression [20, 22]. In the current study, we investigated biomarkers reflecting type VI collagen formation and endotrophin (PRO-C6) as well as type VI collagen degradation (C6M) in a real-world cohort of incident IPF patients enrolled prior to treatment initiation. We showed that type VI collagen formation and to some extent degradation were elevated in patients with progressive IPF compared with stable IPF over 12 months. These results are in line with previous findings from the PROFILE cohort [18, 20]. The present analyses were performed in a real-world cohort, and a less strict definition of progression was used in order to also include patients with marginal physiological progression of disease. Despite using this broad definition of progression, the biomarkers were still elevated in patients with progressive disease similar to previous findings, where a more conservative definition of progression was used. Our study confirms that elevated type VI collagen formation and degradation are associated with progression of IPF, also in early, less advanced disease. In addition, marginal declines in FVC and DLCO have been proven to predict mortality in IPF patients [27, 28]. Also, it has been estimated that the minimal clinically important difference in IPF patients is between 2 and 6% change in % predicted FVC .
We compared longitudinal biomarker levels of PRO-C6 and C6M between untreated patients and the combined treatment group of patients on antifibrotic treatment; the latter had lower values of PRO-C6 and C6M over 12 months compared with the patients without treatment. Furthermore, when we analysed only the progressive patients, the untreated ones had higher levels of C6M; the analyses with only stable patients showed that the untreated group had higher PRO-C6 levels, yet these analyses were limited to a small number of patients of the untreated group. The reason for this difference between the groups is presently unknown and may represent intrinsic differences between the two groups that are not explained by our data or merely due to chance. Patients without treatment were either patients with very mild disease choosing not to start treatment or patients with very advanced disease. Our data could indicate that the majority of patients in the non-treated group have more active disease with higher biomarker levels and a shorter 6MWT distance, yet this is not reflected in the commonly applied physiological variables FVC and DLCO which declined similarly in the treated and untreated groups. Other real-life studies have also reported similar findings . Furthermore, we did not find any differences in the biomarker levels between nintedanib and pirfenidone treated patients, and the PRO-C6 and C6M levels seemed to be stable over 12 months in both groups of patients. This could be due to the relatively less advanced disease of patients in this cohort. It would be interesting to examine PRO-C6 and C6M in a treatment cohort with more advanced disease.
Finally, when we looked at the change from baseline to follow-up in biomarker levels, C6M increased in patients without treatment and remained unchanged in the treated patients. This could indicate that nintedanib and pirfenidone potentially affects the degradation of type VI collagen. Moreover, C6M measures a neoepitope generated by MMP-2 and -9 degradation of type VI collagen  and indirectly it may indicate that nintedanib and pirfenidone could slow down the MMP-2 and -9 activity. In contrast to our finding, it was shown in a small proof of concept study in IPF patients that treatment with omipalisib reduced the levels of PRO-C6 but not C6M . However, this potential antifibrotic treatment has a different mode of action and the study was done in a short time frame compared to PFBIO and this may explain some of the differences.
An important strength of our study is the prospective longitudinal design, in which the collection of blood samples is standardized. Also, we investigate biomarkers in a real-world cohort with less advanced disease comparable to many IPF trial cohorts. On the other hand, a weakness of the current study was that the analyses were only performed in a single cohort with no validation cohort. Furthermore, only few patients progressed when using 10% decline over 12 months, and we, therefore, used a 5% decline to define a progressive phenotype. However, both PRO-C6 and C6M have previously been linked to progression when using the definition of 10% decline in FVC, and the current study confirms that PRO-C6 and C6M are associated with IPF progression and are relevant biomarkers to study further in IPF. The fact that baseline biomarker levels were elevated in untreated patients compared to treated patients was a limitation, and this could affect the interpretation of treatment effects on the biomarkers. We have tried to compensate for this by adjusting for baseline biomarker levels.