Validation and Implementation of OptiView and EnVision FLEX Detection Systems for Immunocytochemical Staining Protocols of the Ten Most Commonly Used Diagnostic Markers in Routine Cytopathological Practice.

The withdrawal of the iView detection system (iV) forced many cytopathology laboratories, including ours, to substitute immunocytochemical (ICC) staining protocols for routine practice with other detection systems. Our objective was to optimize, validate, and implement ICC protocols using OptiView (OV) and EnVision FLEX (EnV) detection systems, comparing the results with those obtained using iV. Residual cytologic samples with known diagnoses were used, testing antibodies for the ten most common markers in routine cytopathology diagnostics (calretinin, Ber-EP4, MOC-31, CKAE1/AE3, CK5/6, CD68, LCA, desmin, HBME-1, and WT1). Different staining parameters were tested using OV on BenchMark ULTRA and EnV on Dako Omnis immunostainer, respectively. Optimal staining protocols were then selected and validated on 10 positive and 10 negative cases. The staining results were compared with iV protocols through evaluation of UK NEQAS and internal scores. The optimal staining protocols with OV and EnV demonstrated similar or superior results compared to the existing iV protocols, with slightly stronger intensity regarding positive cells. We have successfully established and validated optimal ICC staining protocols for commonly used markers in routine cytopathology practice. These protocols may benefit other laboratories using similar staining platforms. However, the challenge regarding standardizing ICC protocols across different cytopathology laboratories remains unresolved.

The prognostic significance of programmed cell death protein 1 and its ligand on lymphoma cells and tumor-immune cells in diffuse large B-cell lymphoma, not otherwise specified.

BACKGROUND: Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) is the most common type non-Hodgkin's lymphoma, where the treatment of relapsed/refractory cases is the major challenge. Programmed cell death protein 1 (PD-1) and its ligand PD-L1 play a crucial role in the negative regulation of the immune response against the disease. The aim of the study was to analyze the expression of PD-1 and PD-L1 on lymphoma cells (LCs) and tumor-immune cells (TICs) and to investigate their correlation with outcome. PATIENTS AND METHODS: Samples from 283 patients diagnosed with DLBCL, NOS (both germinal center B cell like [GCB] and non-GCB subtypes) were included in the study. Expression of PD-1 and PD-L1 was determined using double immunohistochemical staining (D-IHC) for PD-1/PAX5 and PD-L1/PAX5 on tissue microarrays. LCs were highlighted by D-IHC to obtain more accurate results. Clinical data and histologic diagnoses were obtained from electronic data records. We correlated clinical characteristics, and PD-1 and PD-L1 expression on LCs and TICs with progression-free survival (PFS) and overall survival (OS). RESULTS: Expression of PD-1 on TICs was observed in 38.4% and on LCs in 8.8% of cases, while PD-L1 was expressed on TICs in 46.8% and on LCs in 6.5% of cases. PD-L1 expression on LCs was more frequent in non-GCB subtype (p = 0.047). In addition, patients with PD-L1 expression on LCs had significantly shorter PFS (p = 0.015), and the expression retained significant in the multivariate model (p = 0.034). CONCLUSIONS: PD-L1 was more frequently expressed in LCs of the non-GCB subtype. Additionally, PD-L1 in LCs may predict shorter PFS time. D-IHC staining for PD-L1/PAX5 is a feasible method to assess PD-L1 expression on LCs of DLBCL, NOS patients and can be used to identify patients who may benefit from targeted immunotherapy with checkpoint inhibitors.

The Genetic Landscape of Primary Breast Marginal Zone Lymphoma Identifies a Mutational-driven Disease With Similarities to Ocular Adnexal Lymphoma.

Extranodal marginal zone lymphomas (eMZL) can occur in any organ and site of the body. Recent research has shown that they differ from organ to organ in terms of their mutational profile. In this study, we investigated a cohort of primary breast marginal zone lymphomas (PBMZL) to get a better insight into their morphologic and molecular profile. A cohort of 15 cases (14 female and 1 male) was characterized by immunohistochemistry (IHC) for 19 markers, fluorescence in situ hybridization (FISH), and high throughput sequencing (HTS) using a lymphoma panel comprising 172 genes. In addition, PCR for the specific detection of Borrelia spp. and metagenomics whole genome sequencing were performed for infectious agent profiling. Follicular colonization was observed in most cases, while lymphoepithelial lesions, though seen in many cases, were not striking. All 15 cases were negative for CD5, CD11c, and CD21 and positive for BCL2 and pan B-cell markers. There were no cases with BCL2, BCL10, IRF4, MALT1, or MYC translocation; only 1 had a BCL6 rearrangement. HTS highlighted TNFAIP3 (n=4), KMT2D (n=2), and SPEN (n=2) as the most frequently mutated genes. There were no Borrelia spp., and no other pathogens detected in our cohort. One patient had a clinical history of erythema chronicum migrans affecting the same breast. PBMZL is a mutation-driven disease rather than fusion-driven. It exhibits mutations in genes encoding components affecting the NF-κB pathway, chromatin modifier-encoding genes, and NOTCH pathway-related genes. Its mutational profile shares similarities with ocular adnexal and nodal MZL.

Assessment of Ki-67 Proliferative Index in Cytological Samples of Nodal B-Cell Lymphomas.

BACKGROUND: The Ki-67 proliferative index (PI) is part of the diagnosis of nodal B-cell lymphoma (nBCL), but its determination in cytological samples is not standardized. We aimed to establish an approach for the accurate determination of the Ki-67 PI in cytological slides to differentiate between indolent and aggressive nBCLs. METHODS: Patients diagnosed with nBCL by fine-needle aspiration biopsy and subsequent excision biopsy were included. Cell suspensions were prepared from biopsy samples for CD3/Ki-67 double immunocytochemical staining and flow-cytometric verification of lymphoma B-cell counts. The Ki-67 PI was assessed by manual counting and eyeballing in cytology and eyeballing in histology. The cut-off values for the differentiation between aggressive and indolent lymphomas were determined for each method. RESULTS: A strong correlation between manual and flow-cytometric counting of lymphoma B cells was confirmed (interclass correlation coefficient (IC coef.) = 0.78). The correlation of the Ki-67 PI determined in cytological and histological slides was also strong (IC coef. > 0.80). Histologically, 55 cases were classified as indolent and 31 as aggressive nBCLs. KI-67 PI cut-off values of 28.5%, 27.5%, and 35.5% were established for manual counting and eyeballing in cytology and eyeballing in histology, respectively, with high sensitivity and specificity. CONCLUSIONS: The Ki-67 PI, assessed by manual counting and eyeballing in cytological samples, accurately differentiates between indolent and aggressive nBCLs.