Background No plasma biomarkers are associated with the response of acute graft-versus-host disease (GVHD) to therapy after allogeneic hematopoietic stem-cell transplantation. Methods We compared 12 biomarkers in plasma obtained a median of 16 days after therapy initiation from 10 patients with a complete response by day 28 after therapy initiation and in plasma obtained from 10 patients with progressive GVHD during therapy. The lead biomarker, suppression of tumorigenicity 2 (ST2), was measured at the beginning of treatment for GVHD in plasma from 381 patients and during the first month after transplantation in three independent sets totaling 673 patients to determine the association of this biomarker with treatment-resistant GVHD and 6-month mortality after treatment or transplantation. Results Of the 12 markers, ST2 had the most significant association with resistance to GVHD therapy and subsequent death without relapse. As compared with patients with low ST2 values at therapy initiation, patients with high ST2 values were 2.3 times as likely to have treatment-resistant GVHD (95% confidence interval [CI], 1.5 to 3.6) and 3.7 times as likely to die within 6 months after therapy (95% CI, 2.3 to 5.9). Patients with low ST2 values had lower mortality without relapse than patients with high ST2 values, regardless of the GVHD grade (11% vs. 31% among patients with grade I or II GVHD and 14% vs. 67% among patients with grade III or IV GVHD, P<0.001 for both comparisons). Plasma ST2 values at day 14 after transplantation were associated with 6-month mortality without relapse, regardless of the intensity of the conditioning regimen. Conclusions ST2 levels measured at the initiation of therapy for GVHD and during the first month after transplantation improved risk stratification for treatment-resistant GVHD and death without relapse after transplantation. (Funded by the National Institutes of Health.)

A retrospective analysis of 173 skin biopsy specimens of myeloid leukemia cutis (MLC) was performed to determine histologic and immunophenotypic criteria that could distinguish the varied myeloid disorders from one another. For the study, 11 relevant histologic items were scored and 12 antigens were studied (CD68 [KP1], CD163, CD14, CD4, myeloperoxidase [MPO], CD33, CD117, CD34, CD56, MIB-1, CD303, and CD123). Underlying myeloid disorders were essentially acute myeloid leukemias (65.3%), chronic myelomonocytic leukemias (11.0%), and refractory anemia (10.4%). Skin lesions were de novo in 7.5%, concurrent in 26.6%, and subsequent in 60.7%. Several morphologic characteristics (density, size of tumor cells, inflammatory background) were statistically useful in distinguishing between varied myeloid disorders. De novo MLCs displayed a specific morphologic profile. Association of CD68, CD33, and MPO could diagnose 100% of the cases of MLC. However, the immunohistochemical panel could not distinguish between the varied underlying myeloid disorders, with the exception that CD123 was particularly powerful in recognizing chronic myelomonocytic leukemia and also permitted reclassification of 4 cases as blastic plasmacytoid dendritic cell neoplasm.

Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA– transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT.

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2Rα , and REG3α 3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage.For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

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GVHD of the gastrointestinal tract (GI) is associated with high mortality. We have identified systemic, skin- and GI-specific plasma biomarkers present at clinical GVHD onset, and more recently biomarkers for treatment responsiveness. However, in order to identify early GI-specific biomarkers prior to GVHD onset, we performed state-of-the-art proteomics on plasma samples taken 14 days prior to clinical manifestation of GI GVHD. We selected candidates that were increased at least 1.5 fold in plasma from GI GVHD patients compared to HSCT patients without GVHD at matched time points. We identified two lead proteins: CD146, a cell adhesion and trafficking molecule expressed on a subset of CD4+ T cells and endothelial cells, and the chemokine (C-C motif) ligand 14 that binds to the chemokine receptor CCR5 on T cells. As these proteins have not been previously identified in proteomics experiments and antibodies for their corresponding receptors on T cells were available, we analyzed their expression profiles on PB cells from 214 HSCT patients (71 GI GVHD, 48 no GVHD, 33 non-GVHD enteritis, 22 skin first GVHD, 40 isolated skin GVHD) at the onset of symptoms. The frequency of CD146+CCR5+ T cells was significantly increased in GI GVHD patients compared to patients without GVHD, non-GVHD enteritis, or with isolated skin GVHD, as well as increased in patients who first experienced skin and then GI GVHD (Fig. 1). When using the median % of CD146+CCR5+ T cells detected in GI GVHD patients to classify patients into low and high-risk groups, patients in the high-risk group had higher 6-month non-relapse mortality (42 vs. 20%, p = 0.02). Importantly, CD146+CCR5+ T cells at onset of clinical symptoms were not correlated with GI histologic severity, suggesting that these cells are not mucosa damage products but rather systemic effectors. Thus, we measured their frequencies in samples taken at a median of 19 days post-transplant and with a median interval of 14 days prior to clinical symptoms and found that CD146+CCR5+ T cells circulate in patients before GI GVHD clinical onset. In GI GVHD patients, these cells expressed a Th1 and Th17 phenotype and expressed high levels of the activation marker ICOS known to be critical for the development of human Th17 cells. To test the hypothesis that CD146+ T cells are Th17-prone, we next investigated whether in vitro polarization of CD4 T cells with defined stimulation conditions will increase the CD146+CCR5+ expression as well as the production of Th1 and Th17 cytokines. Fig. 2A demonstrates that CD4 T cells differentiated with both Th17-inducing cytokines and ICOS co-stimulation had a significantly higher percentage of CD146+CCR5+ T cells and co-expressed more IL-17A+IFNγ+ than T-cells stimulated with Th1-inducing cytokines or by CD28. We then analyzed colonic mucosa biopsies from patients with GI GVHD (N = 18) and non-GVHD enteritis (N = 10) for the expression of CD146 by immunohistochemistry. CD146 expression was detected on CD3 lymphoid cells and was strongly present on the endothelium. The CD146+ vessel count in GI GVHD tissues was significantly higher than in non-GVHD enteritis tissues (p < 0.001). Th17 cells migrated more efficiently through endothelial cell monolayers than their Th1 counterparts (Fig. 2B). Lentivirus-mediated shRNA knock-down of CD146 in either cell type demonstrated that reduced CD146 expression on CD4+ T cells, but not on endothelial cells significantly reduced the T-cell transendothelial migration (Fig. 2C), suggesting that CD146 on T cells is paramount for promoting infiltration of pathogenic T cells into GVHD target organs. As proof of principle for this hypothesis, we tested donor CD146-/- T cells in an allogeneic murine GVHD model and did not find any difference in GVHD severity when compared to wild type CD146+/+ T cells. Finally, we used a xenogeneic GVHD mouse model with injection of human CD4+ T cells lentivirally transduced with CD146 or control shRNA. In comparison to the vector control group, mice transplanted with CD146 shRNA transduced T cells did not lose weight (Fig. 3A), had similar human T cells engraftment (B), had less splenic CD146+CCR5+ T cells (C), and expressed less TBET 53 days after transplant (D). In conclusion, early quantification of a novel CD146+CCR5+ Th17-prone and ICOS-induced population may allow identification of patients at risk for GI GVHD development and subsequent mortality. Targeting CD146 may represent a new avenue to treat GVHD. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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participate. Overall, 251 pts were identified. At each center, up to 6 pts who started olaparib (400 mg bid, capsule formulation) between 03/2014 and 03/2017 were randomly selected and included. Medical records were reviewed for clinic and pathologic characteristics, survival outcomes and safety. Our primary objective was to assess efficacy of olaparib in real-world pts treated upon initial EMA label (pts EMA ) by evaluating progression free survival (PFS) from olaparib initiation.Results: Overall, 128 pts were included in the analysis and 89 were treated according to EMA label. Main reasons to be given olaparib off-label were absence of radiological response following platinum-based chemotherapy (n¼22) and non high-grade serous EOC subtype (n¼14). BRCA1/2 mutation was present in 126 pts (98%). Most pts (68%) received olaparib after 3 or more lines of platinum-based chemotherapy. Median follow up was 41.8 months. Median PFS in pts EMA was 17.0 months (95% CI: 14.7-21.3). Median PFS and overall survival (OS) in the whole population were 15.5 months (95% CI: 12.6-18.1) and 33.6 months (95% CI: 28.7; 40.3), respectively. Fourteen (11.2%) pts stopped olaparib for toxicity reason and 75 (58.6%) had at least one dose reduction or one dose interruption. Related myelodysplastic syndrome and second cancers were diagnosed in respectively n¼5 and n¼1 pts. Number of previous lines of systemic therapy 2 was associated with prolonged PFS.Conclusions: With an extended follow-up, efficacy and toxicity of olaparib in realworld cohort of pts are consistent with findings observed in study 19 and SOLO-2 trials.Clinical trial identification: NCT04152941.Legal entity responsible for the study: ARCAGY-GINECO.