ECP 2022 Abstract Book

Virchows Archiv (2022) 481 (Suppl 1):S1–S364 13 cell lung carcinoma (NS-NSCLC) patients, notably for a rapid assessment of multiple genomic alterations. Methods: We compared two ultra-fast gene fusion assessment assays, using a next generation sequencing (Genexus, Oncomine™ Precision Assay, Thermo-Fisher) or an RT-PCR (Idylla™, Gen- eFusion Assay, Biocartis) approaches, set up as a reflex testing at diagnosis. Results: 250 NS-NSCLC patients (68 ALK, 26 ROS1, 15 RET, 6 NTRK, 11 MET positive and 125 wild type patients) from 8 centers were included. 83% of patients were stage IIIB-IV. The sensitivity (98%) and specificity (99%) of the two approaches were analogous, when compared to gold standard methods, accred- ited according to the ISO15189 norm in the Laboratory of Clinical and Experimental Pathology (Nice, France). Conclusion: Ultra-fast gene fusion evaluation using NGS or RT-PCR approaches should be developed as a reflex testing for NS-NSCLC at diagnosis in order to treat these patients according to the international recommendations and guidelines. OFP-12-008 Nuclear markers for the diagnosis of histiocytosis I. Ungureanu*, S. Héritier, F. Cohen-Aubart, Z. Hélias-Rodzewicz, J. Donadieu, J. Haroche, J. Emile *Department of Pathology, Ambroise-Paré Hospital, France Background & objectives: Newly described immunomarkers could bring potential benefit in the diagnosis and treatment of histiocytosis. Our objective was to analyse recently described nuclear markers and to expand the immunohistochemical panel in the diagnosis of histiocytosis. Methods: Biopsy samples diagnosed with Erdheim-Chester Disease (ECD), Langerhans cell histiocytosis (LCH), Rosai Dorfman Disease (RDD), malignant histiocytosis, and cutaneous histiocytosis were retrieved from the files of our Pathology Department. Haematoxylin & eosin-stained slides were reviewed. Immunohistochemistry was performed with the following antibodies: PU.1(EPR3158Y, Abcam), OCT.2(EPR12482-106, Abcam), phosphoERK(Erk 1/2, Cell Signalling). Molecular biology was performed using PCR or Next-Generation Sequencing. Results: phosphoERK was performed in 567 biopsy samples of histiocytosis. It was positive in 91%, 86%, 73%, 67%, 70% and 83% of LCH (n=118), ECD (n=198), mixed (n=22), RDD (n=119), cutaneous (n=86) and malignant histiocytosis (n=24), respectively. All the types of histiocytosis were positive for PU.1 (5 LCH, 5 ECD, 7 RDD, 4 ALK+ and 8 C group). Among the 17 cases referred as malignant histiocytosis, all the 9 confirmed cases were positive, contrasting with the 8 excluded cases that were negative. OCT.2 was positive in 42/80 cases of RDD. All 9 mixed RDD-ECD or RDD-LCH were OCT.2 positive. 18/38 cases of non-RDD had at least a low positivity for OCT.2. Conclusion: PU.1 is a marker indicating a histiocytic origin and it is useful to exclude a tumour rich in reactive histiocytes mimick- ing histiocytosis. OCT.2 can be used to confirm Rosai-Dorfman Disease. phosphoERK is a nuclear and cytoplasmic marker high- lighting the activation of MAPkinase pathway and it can be used to initiate targeted therapy in histiocytosis when molecular biology is not available. OFP-12-009 Clonality analysis of Richter’s transformation in CLL treated with targeted therapy I. Xie*, I. Rashedi, Y. Amemiya, D. Spaner, A. Seth, Z. Ghorab, R. Goswami *Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada Background & objectives: Richter’s syndrome (RS) occurs in 1-10% of patients with chronic/small lymphocytic leukaemia (CLL/ SLL). Clonal transformation has a poorer prognosis than de novo transformation, and benefits from more aggressive chemotherapy. We sought to characterize RS clonality in a Canadian case series. Methods: DNA was extracted from FFPE lymph node, bone marrow, or banked cells. Targeted NGS of heavy and light chain regions was performed using the Oncomine BCR Pan-Clonality Assay (ThermoFisher). IGH, IGK, and IGL rearrangements and clonal frequencies were assessed through the Ion Reporter Oncomine BCR workflow and MiXCR. Slides and IHC were reviewed to confirm the diagnosis. Results: We identified nine patients with 20 pre- and post- transformation samples. The median age at diagnosis was 52 (range 44-84), with median 9 years (range 1.9-15) between the diagnosis of CLL vs RS. Most cases transformed to DLBCL (n=7), and the remainder to classical Hodgkin lymphoma (n=2). 7/9 patients had received therapy prior to transformation, including ibrutinib, acalabrutinib, or venetoclax. Analysis of the heavy chain and light chain loci revealed a clonal relationship in all nine cases, including one case where the secondary DLBCL was CD5-negative. Interestingly, several cases exhibited multiple productive IGH rearrangements (2/9) or light chain rearrangements (7/9) shared between the pre- and post-transformation cells. Conclusion: Targeted sequencing of a case series of CLL patients with RS revealed a clonal relationship in all nine cases. Multiple productive Ig rearrangements were identified in a surprising number of cases, consistent with a growing number of NGS studies support- ing greater diversity in CLL clonality than previously appreciated. OFP-12-010 Delineating the spatial compartmentalization of human follicular B cell metabolic dynamics M.M. Perra*, A. Noto, K. Ioannidou, M. Foglierini-Perez, C. Fen- wick, L. de Leval, C. Petrovas, G. Pantaleo *Unil-CHUV, Switzerland Background & objectives: Despite the well-established role of germinal centers (GC) for the generation of protective B cell response, an insight on the in-situ human GC immune reactions remains unclear. We aimed to evaluate the energetic and metabolic profile of follicular B cells. Methods: Metabolic profile of primary tonsillar B cells were char- acterized by applying complementary methodologies. Phenotyping and 2-NBDG uptake of relevant B cell subsets were assessed by multiparametric flow-cytometry. Ex vivo metabolic function (Sea- horse) and transcriptomic signatures (bulk NGS) were investigated on cryopreserved TNMCs (tonsillar mononuclear cells). Multiplex tissue imaging was applied for the in situ B cell metabolic profiling. Results: Seahorse analysis revealed that OXPHOS supported 70% and glycolysis 30% of total ATP produced in GCBCs. Coherently, an increased 2-NBDG uptake was observed in GCBCs. GSEA of sorted B-cell subsets showed significant enrichment of OXPHOS and mTORC1 pathways in GCBCs, with several glycolysis-related genes being significantly expressed. In-vitro treatment with UK5099 (MPC inhibitor), negatively affected the SRC, MRC, and Krebs capacity in GCBCs as compared to untreated controls. Tis- sue imaging of FFPE-tonsillar sections revealed a polarization of GLUT1, MCT4, HIF1a and HIF1b in Light Zone, while Opa1 was expressed within Dark Zone. Gene expression of MCT1 (lactate transporter) was increased in GCBCs, while imaging showed its distribution across the GC. S51