Objective:To detect the expression of sialylated CD15 (CD15s) in the tumor cells of classical Hodgkin lymphoma using a modified immunohistochemical approach.Methods:From 2009 to 2024, 53 cases of classical Hodgkin lymphoma were collected in the Department of Pathology, Peking University Cancer Hospital, in which 21 cases that were CD15-negative or showed only focal weak positivity were selected. Immunohistochemical staining for CD15 was performed on a Leica automated stainer using three different antibody clones (MMA, Carb3, and IHC527). Tissue sections were digested with sialidase at varying concentrations and incubation times, followed by immunohistochemical staining with the MMA clone. Multiplex immunofluorescence was applied for co-staining of CD15 (MMA) and CD30 (JCM182), and analysis was conducted using APTIME and HALO software.Results:There were 30 male patients and 23 female patients, with an age range of 14 to 73 years and a median age of 32(26,46) years. None of the three CD15 antibody clones significantly improved the CD15 positive rate in the 14 completely negative and 7 weakly positive cases, with no notable differences observed among the clones( P>0.05). After sialidase digestion, tissue morphology remained well-preserved. Optimal CD15 staining was achieved with a 1∶1 diluted sialidase incubated at 37 ℃ for one hour. This treatment significantly enhanced the detection rate of CD15 antigen in Hodgkin Reed-Sternberg cells ( P<0.001). Conclusion:Sialidase digestion effectively unveils sialylated CD15 expression in classical Hodgkin lymphoma, markedly improving its detection in HRS cells.

Objective:To explore the values of reticulin fiber staining (RFS) in evaluating bone marrow (BM) involvement of lymphoma and in grading of BM biopsy from adult lymphoma patients.Methods:Retrospectively,354 cases of adult lymphoma were collected from November 2023 to May 2024 at Peking University Cancer Hospital. BM samples were stained with RFS and immunohistochemical staining (IHC), and flow cytometry (FCM) was also performed with the BM aspirations simultaneously. RFS was graded according to the European Consensus, as high grade (grade 2-3) indicating BM involvement in the study. BM involvement was considered as definite if no less than two positive findings among IHC, FCM, and RFS. Statistical analyses were performed via SPSS software (V23.0).Results:In this series, 52.3% (185/354) of the patients were male; 35.0% (124/354) aged >60 years; BM involvements were found in 34.5% (122/354) cases with high grade of RFS, which, in turn, were lymphoblastic leukemia/lymphoma (ALL/LBL) group (4/4), indolent B-cell lymphoma (IndBCL) group (49.1%, 53/108), transformed B-cell lymphoma (TrBCL) group (2/5), invasive B-cell lymphoma (InvBCL) group (26.5%, 41/155), T and NK cell lymphoma (TNKCL) group (27.3%, 12/44) and classical Hodgkin lymphoma (CHL) group (26.3%, 10/38); if classified by specific types, T-ALL/LBL (2/2), B-ALL/LBL (2/2) and CLL/SLL (8/10) ranked top three. In terms of the positive rate of BM involvement evaluated by RFS, no significant difference was seen between either gender or age groups ( χ2=3.416, P=0.332 and χ2=4.200, P=0.241); however, significant differences were observed between different lymphoma groups and types ( χ2=29.961, P=0.012 and χ2=102.546, P<0.001, respectively). BM invasion rates indicated by IHC and FCM were 25.4% (90/354) and 13.8% (49/354), respectively. The overall BM invasion rate was 24.3% (86/354), and the sensitivity of RFS, IHC, and FCM was 90.8%, 97.8%, and 55.8%, and specificity was 84.1%, 99.6%, and 98.9%, respectively. Overall, the concordance rate of RFS with IHC and FCM was 83.6% and 74.0%, respectively, including 85.8% and 74.2% for InvBCL group, 79.6% and 75.0% for IndBCL group, 84.1% and 75.0% for TNKCL group, 81.6% and 73.7% for CHL group, 5/5 and 2/5 for TrBCL group, and 4/4 and 3/4 for ALL/LBL group. Conclusions:In the evaluation of BM involvement status of adult lymphoma, high sensitivity and specificity are observed by RFS, and high concordance is also noted with both IHC and FCM. Thus, the BM infiltrating status of adult lymphoma could be evaluated more accurately by a combined usage of the three methods.

Objective:To explore the values of reticulin fiber staining (RFS) in evaluating bone marrow (BM) involvement of lymphoma and in grading of BM biopsy from adult lymphoma patients.Methods:Retrospectively,354 cases of adult lymphoma were collected from November 2023 to May 2024 at Peking University Cancer Hospital. BM samples were stained with RFS and immunohistochemical staining (IHC), and flow cytometry (FCM) was also performed with the BM aspirations simultaneously. RFS was graded according to the European Consensus, as high grade (grade 2-3) indicating BM involvement in the study. BM involvement was considered as definite if no less than two positive findings among IHC, FCM, and RFS. Statistical analyses were performed via SPSS software (V23.0).Results:In this series, 52.3% (185/354) of the patients were male; 35.0% (124/354) aged >60 years; BM involvements were found in 34.5% (122/354) cases with high grade of RFS, which, in turn, were lymphoblastic leukemia/lymphoma (ALL/LBL) group (4/4), indolent B-cell lymphoma (IndBCL) group (49.1%, 53/108), transformed B-cell lymphoma (TrBCL) group (2/5), invasive B-cell lymphoma (InvBCL) group (26.5%, 41/155), T and NK cell lymphoma (TNKCL) group (27.3%, 12/44) and classical Hodgkin lymphoma (CHL) group (26.3%, 10/38); if classified by specific types, T-ALL/LBL (2/2), B-ALL/LBL (2/2) and CLL/SLL (8/10) ranked top three. In terms of the positive rate of BM involvement evaluated by RFS, no significant difference was seen between either gender or age groups ( χ2=3.416, P=0.332 and χ2=4.200, P=0.241); however, significant differences were observed between different lymphoma groups and types ( χ2=29.961, P=0.012 and χ2=102.546, P<0.001, respectively). BM invasion rates indicated by IHC and FCM were 25.4% (90/354) and 13.8% (49/354), respectively. The overall BM invasion rate was 24.3% (86/354), and the sensitivity of RFS, IHC, and FCM was 90.8%, 97.8%, and 55.8%, and specificity was 84.1%, 99.6%, and 98.9%, respectively. Overall, the concordance rate of RFS with IHC and FCM was 83.6% and 74.0%, respectively, including 85.8% and 74.2% for InvBCL group, 79.6% and 75.0% for IndBCL group, 84.1% and 75.0% for TNKCL group, 81.6% and 73.7% for CHL group, 5/5 and 2/5 for TrBCL group, and 4/4 and 3/4 for ALL/LBL group. Conclusions:In the evaluation of BM involvement status of adult lymphoma, high sensitivity and specificity are observed by RFS, and high concordance is also noted with both IHC and FCM. Thus, the BM infiltrating status of adult lymphoma could be evaluated more accurately by a combined usage of the three methods.

Objective:To detect the expression of sialylated CD15 (CD15s) in the tumor cells of classical Hodgkin lymphoma using a modified immunohistochemical approach.Methods:From 2009 to 2024, 53 cases of classical Hodgkin lymphoma were collected in the Department of Pathology, Peking University Cancer Hospital, in which 21 cases that were CD15-negative or showed only focal weak positivity were selected. Immunohistochemical staining for CD15 was performed on a Leica automated stainer using three different antibody clones (MMA, Carb3, and IHC527). Tissue sections were digested with sialidase at varying concentrations and incubation times, followed by immunohistochemical staining with the MMA clone. Multiplex immunofluorescence was applied for co-staining of CD15 (MMA) and CD30 (JCM182), and analysis was conducted using APTIME and HALO software.Results:There were 30 male patients and 23 female patients, with an age range of 14 to 73 years and a median age of 32(26,46) years. None of the three CD15 antibody clones significantly improved the CD15 positive rate in the 14 completely negative and 7 weakly positive cases, with no notable differences observed among the clones( P>0.05). After sialidase digestion, tissue morphology remained well-preserved. Optimal CD15 staining was achieved with a 1∶1 diluted sialidase incubated at 37 ℃ for one hour. This treatment significantly enhanced the detection rate of CD15 antigen in Hodgkin Reed-Sternberg cells ( P<0.001). Conclusion:Sialidase digestion effectively unveils sialylated CD15 expression in classical Hodgkin lymphoma, markedly improving its detection in HRS cells.

BACKGROUND: The expression of programmed cell death ligand 1 (PD-L1) as a biomarker for immunotherapy in non-small cell lung cancer (NSCLC) is routinely detected in clinical pathology department. However, the spatial heterogeneity of PD-L1 expression in intrapulmonary tumors and extrapulmonary metastases is still a challenge for the clinical testing. This study aims to explore the differences of PD-L1 expression in test samples obtaining from different sites of NSCLC. This study may contribute to the detection strategy of PD-L1 in patients with advanced lung cancer. METHODS: One hundred and thirty-one cases of consecutively detected PD-L1 (22c3 assay, Dako) staining in metastatic NSCLC and 972 cases of non-paired intrapulmonary NSCLC were collected. The discrepancies of tumor proportion score (TPS) of PD-L1 expression in intrapulmonary samples and extrapulmonary metastatic samples of different sites were compared. RESULTS: The positive expression rate of PD-L1 in extrapulmonary metastatic NSCLC (TPS ≥ 1%) was 61.83%, and the TPS was significantly higher than that in intrapulmonary tumors (P=0.03). The PD-L1 scores of the specimens obtained from different sites were significantly different (P=0.007). The positive rates of PD-L1 in liver and adrenal metastases were 85.71% and 77.78% respectively, and their TPS were significantly higher than that of the intrapulmonary samples (P<0.05). The positive rates of PD-L1 in lymph node, bone, brain, soft tissue, and pleural metastases was 40.00%-66.67%, with no significant differences compared to intrapulmonary tumors. The analysis of histological subtype and sample type showed that the PD-L1 score of extrapulmonary samples of adenocarcinoma subtype or surgical specimen was significantly higher than that of intrapulmonary tumors. The analysis of clinicopathological parameters showed that the PD-L1 positive expression or high expression were significantly correlated with male patients, smoking history, and epidermal growth factor receptor (EGFR) wild type. CONCLUSIONS The expression of PD-L1 in metastatic NSCLC is generally higher than that in intrapulmonary tumor, and the positive rate of PD-L1 expression was discrepant in different sites of specimen. The differences of PD-L1 score between extrapulmonary metastatic samples and intrapulmonary samples may be associated with different metastatic sites, histological subtype, and specimen type.
B7-H1 Antigen/metabolism* ; Biomarkers, Tumor/metabolism* ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Humans ; Immunohistochemistry ; Lung Neoplasms/drug therapy* ; Male