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Supplementary information and data
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Supplementary information- ROC curve analysis
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The area under the ROC curve (AUC) is a measure of the discriminatory power of the test and is
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closely related to the Mann-Whitney U test; a perfect test would give an area of 1 while an
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insensitive test would give an area of 0.5. A cut-off point is required to optimise the ratio of false
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positives to false negatives. This point was determined for each biomarker by minimising the
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errors while disregarding relative costs associated with incorrect positive/negative assignment (1).
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In this test the true positive rate (Sensitivity) is plotted against the false positive rate (100-
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Specificity) for different cut-off points of a parameter. Each point on the ROC curve represents a
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sensitivity/specificity pair corresponding to a particular decision threshold. The area under the
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ROC curve is a measure of how well a parameter can distinguish between two diagnostic groups
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(diseased/normal). An estimate of the pre-test disease prevalence (proportions of subjects who
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do/do not have the disease) was determined from the population that underwent HSCT at the
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Newcastle and Regensburg centres between 11/02/2002 and 18/02/2009, (n=339). Clinically, the
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disease prevalence is the same as the probability of disease being present before the test is
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performed. This estimate (0.6) was used to determine the cut-off point for each biomarker for both
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aGVHD and cGVHD and is determined from the point on the respective curve where the slope is
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equal to the ratio of disease- negative to disease- positive patients in the population P(D-)/P (D+)
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(2).
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Supplementary information- Skin explant model
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Briefly, recipient skin biopsies and blood were taken pre-transplant. Recipient skin was co-
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cultured with mixed lymphocyte reaction (MLR) primed donor allo-reactive T cells. Skin sections
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were then paraffin embedded, stained and graded for histopathological damage using the Learner
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scoring criteria, grades 0-IV (3). Grade 0 or I represents a negative response with an intact upper
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keratinocyte layer. A grade II> response represents a positive response and shows vacuolisation
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of keratinocytes. Grade III response shows formation of clefts and the beginning of separation of
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the dermis/epidermal layer. Grade IV shows complete separation of the dermis and epidermal
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layers. Negative controls consisted of incubation of recipient skin biopsy with autologous cells or
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in medium alone.
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Supplementary data- Effect of conditioning regimens on CXCL10 and CXCL11 levels
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compared to CXCL10 levels in normal healthy individuals. CXCL10 levels in HSCT patients prior
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to conditioning (pre) were similar to levels in normal healthy individuals. However, CXCL10
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levels significantly increased in HSCT patients receiving myeloablative or RIC treatment when
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compared to normal healthy individuals (myeloablative; day 0 p=0.04, day14 p=0.0007, day28
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p=0.002, 3 months p=0.0005, 6 months p=0.001 and 12 months p=0.002) (RIC; day 0 p=0.007,
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day 14 p=0.001, day 28 p=0.0006, 3 months p=0.01, 6 months p=0.003 and 12 months p=0.007)
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(Figure 1A) and when compared to pre-transplant levels (myeloablative; day14 p=0.007 day28
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p=0.0003, 3 months p=0.004, 6 months p=0.06 and 12 months p=0.06) (RIC; day 0 p=0.0002, day
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14 p=0.0001, day 28 p=0.0001, 3 months p=0.005, 6 months p=0.02 and 12 months p=0.01) .
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When CXCL10 levels were compared between the two conditioning treatments no statistical
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difference was observed between patients undergoing myeloablative or RIC treatment at each
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time-point. CXCL11 levels were significantly increased in HSCT patients undergoing
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myeloablative or RIC treatment when compared to normal healthy individuals (myeloablative; day
CXCL10 levels in NCL HSCT patients who received either myeloablative or RIC treatment were
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0 p=0.005, day14 p=0.04, day28 p=0.02, 3 months p=0.03 and 6 months p=0.03) (RIC; day 0
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p=0.004, day 14 p=0.01, day 28 p=0.0007, 3 months p=0.01, 6 months p=0.009 and 12 months
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p=0.005) (Figure 1B). No difference was observed in CXCL11 levels between pre-transplant
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levels and post-transplant in myeloablative patients. However, a significant increase was observed
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in RIC patients at day 0 and day 28 (p=0.009 and p=0.0006, respectively) compared to pre-
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transplant levels. No statistical difference in CXCL11 levels between the two conditioning
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treatments was observed.
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References
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