Biological Monitoring of Extracorporeal Photopheresis for Steroid Resistant Acute Graft versus Host Disease by a Short Culture Method and Flow Cytometry

Graft versus host disease is a major complication of allogeneic hematopoietic stem cell transplantation , occurring as acute and severe form in around 15% of patients; in the chronic form GVHD is the main cause of non relapse mortality in patients surviving more than 2 years from transplant [1] . The first line treatment of both severe aGVHD and persisting cGVHD consists of steroids. On steroid treatment, around 60 % of patients with aGVHD fails to respond (steroid-refractory; SR), showing a long-term survival of 5-30% [2] . Second line treatments for SR aGVHD includes extracorporeal photopheresis (ECP) [3] . It has been reported in prospective studies that ECP produces a multi-organ response in SR aGVHD, ranging from 55 % to 84% [4]. Some recent guidelines report indications, methods and schedule for the treatment of SR aGVHD patients by ECP [5,6] . The other second line treatments of SR aGVHD, such as anti-cytokine and other immunosuppressant drugs, have a quite clear mechanism of action, while ECP action is not completely understood. Nevertheless, ECP action seems to be related to a sequence of cascade phenomena, starting with reinfusion of patients lymphocytes induced to apoptosis and ultimate death, followed by their phagocytosis by dendritic cells (DC), production of anti-inflammatory molecules, induction of DC maturation and Th2 phenotype in T cells and , finally, by generation of T regulatory cells [7] . Thus, the primary consequence of ECP treatment consists of the reinfusion of T cells that have been induced to apoptosis/death, promoting T cell regulation and immune tolerance. Recently, a simple and functional method to assess apoptosis in T cells after ECP has been proposed. This method is based on leukocyte isolation from buffy coat obtained by ECP, 48-72 hour cultures of leukocyte in growth medium and subsequent cytofluorimetric analysis to detect CD3+ cell apoptosis/death using Annexin V/7-Amino-Actinomycine D (7-AAD) staining [5,8] . This test has been proposed as quality control to check the first ECP procedures and represents a sort of biological validation, to be repeated after 8-Methoxy-Psoralen (8-MOP) /disposable lot changes or other procedure variations, including UVA source change. In the present report, we have analyzed results of CD3+ cell Annexin V/7-AAD staining in 29 patients who underwent ECP for grade 3/4, SR aGVHD.

ECP was performed in 29 patients for a total of 725 procedures with a median number of 21 procedures per patient. We obtained a leukocyte concentrate of 157 mL +/- 28 mL on average, processing a patient blood volume ranging from 1,500 ml to 2,000 ml using the Cellex Therakos closed system. The first procedure of ECP was subjected to quality control by Annexin V/7-AAD cytofluorimetric analysis of collected CD3+ cells, prior and after irradiation and following a 48-hour culture. The quality control was repeated during the second ECP as replicate confirmation. In our series we observed three different sample staining patterns as follows : 1) relevant induction of cell apoptosis/death with Annexin V and or 7-AAD staining increase > 40 % as compared to the non-irradiated control (defined as “positive result”; Figure 1); 2) cell apoptosis/death with Annexin V and or 7-AAD staining increase < 40 % (defined as “negative result”); 3) no relevant changes between non-irradiated and irradiated CD3+ cells after culture (this pattern always showed high level of cell apoptosis/death phenomena prior and after cell irradiation, defined as “no change”). Overall, most patients experienced relevant apoptotic phenomena as compared to PreECP conditions [72 % had > 40 % Annexin V and/or 7-AAD staining difference ) on CD3+ cells, prior and after irradiation; Table 1) at the time of first ECP procedure. We observed a parallel increase in Annexin V and 7-AAD fluorescence even though, in 4 cases out of 21, positivity for Annexin V paralleled a border-line 7-AAD staining (increase of 7-AAD staining was around 40 %). In eight cases we observed 3 “negative result” and 5 “no change”. Table 1 shows that lymphocyte count/μL in collected buffy coat was extremely variable, ranging from 360 to 23,100. We didn’t find a significant correlation between the lymphocyte count in the buffy coat and a “positive result” of Annexin V/7-AADΔ (R2=0.027; p= 0.393). No correlations were found between age, gender, disease, aGVHD grade and time from transplant to first ECP and Annexin V/7-AAD Δ (p>0.05 in all cases). We built a score, ranging from 0 to 2 (see methods), which combines survival on aGVHD and the number of ECP procedures performed in each patient (< 21 procedures, > 21 procedures); this score was challenged at linear regression analysis with a “positive result” for Annexin V/7-AADΔ and we found a weak, albeit significant, correlation (R= 0.395; p=0.033). This score correlated neither with lymphocyte count in the buffy coat, nor with gender , disease, age, aGVHD grade and the number of days between transplant and the first ECP. To ascertain the influence of possible confounding effects, we performed a multiple regression analysis between score and Annexin V/7-AAD lymphocyte count in the buffy coat, gender, disease, age, aGVHD grade and the number of days from transplant to the first ECP. By this additional analysis Annexin V/7-AADΔ maintained its statistical significance (Coefficient 0.952, p=0.004; Table 2).

In patients who had undergone allogeneic stem cell transplantation aGVHD is the main cause of non-relapse mortality. Sixty percent of them do not respond to the standard treatment based on steroids. Once steroid refractory (SR), these patients need a second-line treatment and ECP is an effective option to restore immune tolerance of the allograft [1-6]. ECP, through the effect of 8-MOP and UVA light, produces changes in patients’ collected lymphocytes and other cell lineages that, ultimately, translate into a regulatory behavior of the immune system [7]. The Cellex photopheresis system is a dedicated technology that allows collection, 8-MOP exposure and UVA irradiation of patients’ MNC in a closed system by an apheretic session lasting around 3 hours [9]. Several recent guidelines indicate ECP as a good option for second line treatment of SR aGVHD, with a procedure schedule based on 2 consecutive procedures per week and subsequent tapering, performing 2 procedure any other week, on the basis of careful evaluation of organ response and of current steroid dosage [5,6] . The present analysis reports the performances of a previously described ECP quality control, based on lymphocyte culture and flow cytometric staining for Annexin V and 7-AAD [8] . This test had been proposed as a quality check for ECP to confirm the induction of CD3+ cells apoptosis/death after irradiation since no other simple markers to monitor in vitro efficacy of this procedure are available [5]. Our series of 29 patients were monitored with flow cytometric monitoring of Annexin V/7-AAD Δ on irradiated CD3+ cells on the first ECP, with a confirmatory replicate test during the second consecutive ECP. In the present report we observed a good performance of this quality test that gaves the expected result in more than 70 % of patients, and a “negative result” or a “no change “ result in the rest of patients (see results and figure 1). In the “no change” pattern (5 patients) a relevant apoptosis/death phenomena was observed in the non-irradiated control with a marginal increase induced by ECP. This pattern represents a quite unexplored biological event, at least in these patients, and, as hypothesis, could be related to a massive and pre-existing lymphocyte behavior, en route to death, because of the powerful lympholytic activity of immunosuppressive drugs. In this case, the contribution of ECP could be questionable and should deserve further investigations. In only 3 cases we found a “negative result”, consisting in the inability of CD3+ cells to start a death program in spite of irradiation by ECP. These results were confirmed in the replicate confirmatory tests after the second ECP and could be related to a resistance of individual lymphocytes to activate a death program , at least in the standard conditions by which we perform ECP . Indeed, we did not analyze the subsets of CD3+ cells in cases of “no change” or negative results, in order to understand if resistance or hyper-sensitivity to cell death were caused by the prevalence of a specific immune subset. Annexin V/7-AADΔ analysis was statistically correlated to a score which explores the patients’ outcome after ECP and we found a significant result at simple and multiple regression analyses, being age, disease, gender, aGVHD grade, lymphocyte count in the BC and time from transplant to ECP never significant. As to the role of lymphocyte count in the BC, a recent report found a positive correlation between the dose of irradiated and reinfused lymphocyte per kg of body weight per procedure and ECP response at 1 month [10] . On the other hand, our analysis evaluated only the role of lymphocyte concentration in the first ECP and not the dosage per kg of body weight, likely explaining the variance with results of Worel et al [10] . Overall, our study indicates the following circumstances:

Finally, our study shows interesting suggestions using an in vitro quality control of ECP and encourages its introduction in more centers to verify in large patients’ series its real prediction ability. Unfortunately, the functional nature of this test could prevent the routine use in most centers, as surveyed in a recent report [11] .Centralization of this quality control could be a good chance for standardization and to widen its use for ECP in vitro monitoring.