## 4 Discussion

Table 5: Odd ratio and risk ratio for oGVHD onset of the difference pre-post-HSCT of tear total protein (TP), latorferrin (LACTO), transferrin (TRANSF), and zinc-alpha-2-glycoprotein (ZAG-2). Currently, almost 1800 proteins are known to constitute the human tear proteome, and some panels for up or down-regulation in DED have been recently proposed [44]. To briefly review tear proteins analyzed in this paper, tear LACTO along with LYS-C and LIPOC-1 are co-localized with in the lachrymal gland secretory granules, they might most likely be secreted together, suggesting a role as indicators for the lachrymal gland function [45]. In particular, LACTO and LYS-C are multifunction chain polypeptides with anti-inflammatory, bacteriostatic, and antioxidant properties, whereas LIPOC-1 is the main lipid carrier and scavenger in human tears and is crucial in the ocular surface protection [46; 47; 45]. A recent meta-analysis suggested LACTO level in tears is a good candidate as dry eye syndrome diagnostic biomarker [48]. The plasma-derived proteins ALB and TRANSF can be considered as indirect sign of subclinical inflammation as it occurs when there is an increase of protein leakage from inflamed conjunctival vessels [49; 50; 51]. They are usually present in a negligible amount in tears of normal subjects [26], and it has been shown that ALB increases whereas TRANSF decreases in moderate DED [27]; it is not clear why these proteins behave contrarily and if this may be due to blood-epithelial barrier functioning. First reported in human serum and later in other body fluids, ZAG-2 represents an interesting protein that stimulates lipid breakdown in adipocytes, and which is highly expressed in cancer cachexia [52]. A reduction of ZAG-2 tear levels was documented in CL wearers [53], DED [27; 54], fungal infections at an early stage [55; 56; 57], diabetic patients [58], and an increase in tears of smokers and patients with [PERSON]'s ophthalmopathy [59]. The function of ZAG-2 in tears and its possible role in lipid degradation has been postulated but not demonstrated [59]. A decrease of TP levels in the tears may generally indicate a reduction in synthesis capability by the lachrymal glands, as it has been hypothesized in [PERSON]'s patients [32]. Lacrimal gland dysfunction and fibrosis is a major feature of oGVHD [60], and this may explain the decreased level of the tear TP in our patients, with distinctive profiles identified in this longitudinal study earlier than the clinical diagnosis. Some results were in agreement with what was found previously in post-HSCT patients who had developed oGVHD [18; 19], in particular the reduction of LACTO and LYS-C, whereas another major tear proteins such as LIPOC-1 appeared unmodified in our study. We hypothesize this might be related to the tear analysis performed early after HSCT, with oGVHD yet to come, and with a lachrymal gland still partially functioning and secreting LIPOC-1. Increased levels of plasma-derived TRANSF but not ALB might indicate an early modification of blood-epithelial barrier, and as above reported a sign of exudation and inflammation at a subclinical level. Lowering of tear ZAG-2 in this study is unexpected, as these are cancer-suffering patients, where an increase of ZAG-2 plasma levels had been observed. The pre-post change of this protein seems to be highly correlated with the oGVHD development, so the findings from this work are a preliminary step to deepen ZAG-2 role in this progression. Taken as a whole, TRANSF and ZAG-2 changes are indicative of inflammation and lipid metabolism alteration, earlier than oGVHD onset. Parameters of ROC curves, in particular the AUC, for TRANSF and ZAG-2 appear to be less significant than those reported in [18], but in this paper the diagnostic accuracy for oGVHD had been analyzed versus healthy controls.