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.

Morphological and Immunocytochemical Characterization of Tumor Spheroids in Ascites from High-Grade Serous Carcinoma.

Tumor spheroids in the ascites of high-grade serous carcinoma (HGSC) are poorly described. Our objective was to describe their morphological features, cellular composition, PD-1 and PD-L1 expression, and survival correlation of these parameters. The density and size of spheroids were assessed in Giemsa-stained smears; the cell composition of spheroids, including tumor cells, immune cells, capillaries, and myofibroblasts, as well as PD-1 and PD-L1 expression on tumor and immune cells was assessed in immunocytochemically stained cell block sections. Forty-seven patients with primary HGSC and malignant ascites were included. A cut-off value for a spheroid density of 10% was established, which significantly predicted overall survival. However, spheroid size did not correlate with survival outcomes. Spheroids were primarily composed of tumor cells, but the presence of lymphocytes and macrophages was also confirmed. Moreover, capillaries were present in the spheroids of three patients, but the presence of myofibroblasts was not confirmed. PD-1 was expressed on lymphocytes but not on tumor cells. PD-L1 expression was seen on both tumor and immune cells, assessed by 22C3 and SP263 antibody clones but not by the SP142 clone. Our results highlight the potential of routine cytopathological techniques to analyze spheroids in HGSC ascites as a valuable tool to investigate their potential as prognostic markers.

The Prognostic and Predictive Value of Human Gastrointestinal Microbiome and Exosomal mRNA Expression of PD-L1 and IFNγ for Immune Checkpoint Inhibitors Response in Metastatic Melanoma Patients: PROTOCOL TRIAL.

BACKGROUND: Immunotherapy has been successful in treating advanced melanoma, but a large proportion of patients do not respond to the treatment with immune checkpoint inhibitors (ICIs). Preclinical and small cohort studies suggest gastrointestinal microbiome composition and exosomal mRNA expression of PD-L1 and IFNγ from the primary tumor, stool and body fluids as potential biomarkers for response. METHODS: Patients treated with immune checkpoint inhibitors as a first line treatment for metastatic melanoma are recruted to this prospective study. Stool samples are submitted before the start of treatment, at the 12th (+/-2) week and 28th (+/-2) week, and at the occurrence of event (suspected disease progression/hyperprogression, immune-related adverse event (irAE), deterioration). Peripheral venous blood samples are taken additionally at the same time points for cytologic and molecular tests. Histological material from the tumor tissue is obtained before the start of immunotherapy treatment. Primary objectives are to determine whether the human gastrointestinal microbiome (bacterial and viral) and the exosomal mRNA expression of PD-L1 and IFNγ and its dynamics predicts the response to treatment with PD-1 and CTLA-4 inhibitors and its association with the occurrence of irAE. The response is evaluated radiologically with imaging methods in accordance with the irRECIST criteria. CONCLUSIONS: This is the first study to combine and investigate multiple potential predictive and prognostic biomarkers and their dynamics in first line ICI in metastatic melanoma patients.

Association of Circulating Tumor Cells, Megakaryocytes and a High Immune-Inflammatory Environment in Metastatic Breast Cancer.

Liquid biopsy is becoming an important source of new biomarkers during the treatment of metastatic cancer patients. Using size-based microfluid technology, we isolated circulating tumor cells (CTCs) from metastatic breast cancer patients to evaluate their presence and cluster formation, as well as the presence of megakaryocytes and immune-inflammatory blood cells, and to correlate their presence with clinicopathological data and overall survival (OS). In total, 59 patients (median age 60.4 years) were included in the study: 62.7% luminal A/B-like, 20.3% HER2-positive, and 17% triple-negative. Our results showed that at least one CTC was present in 79.7% and ≥5 CTCs in 35.2% of the patients. CTC clusters were present in patients with ≥5 CTCs only (in 19.2% of them), and megakaryocytes were present in 52% of all patients. The presence of CTC clusters and megakaryocytes was positively associated with the CTC count. Patients with low pan-inflammatory value (PIV), low systemic immune-inflammatory index (SII), and low relative change from baseline (ΔPIV%, ΔSII%) were associated with significantly higher OS than their counterparts. ΔPIV%, the presence of infection in the last month, and a long duration of metastatic disease were identified as independent prognostic factors for OS. The interplay of CTCs, CTC clusters, megakaryocytes, and PIV needs to be further explored.

CD56-positive diffuse large B-cell lymphoma: comprehensive analysis of clinical, pathological, and molecular characteristics with literature review.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. The expression of CD56 in DLBCL is highly unusual. Little is known about its incidence and clinical importance. So far, no genetic profiling was performed in CD56 positive DLBCL. PATIENTS AND METHODS: Tissue microarrays have been constructed, sectioned, and stained by H&E and immunohistochemistry for 229 patients with DLBCL diagnosed 2008-2017. For CD56 positive cases, clinical data was collected including age at diagnosis, stage of the disease, International Prognostic Index (IPI) score, treatment scheme and number of chemotherapy cycles, radiation therapy, treatment outcome, and possible relapse of the disease. Overall survival (OS) and progression-free survival (PFS) were calculated. For four patients, RNA was extracted and targeted RNA (cDNA) sequencing of 125 genes was performed with the Archer FusionPlex Lymphoma kit. RESULTS: CD56 expression was found in 7 cases (3%). The intensity of expression varied from weak to moderate focal, to very intensive and diffuse. All patients had de novo DLBCL. The median age at the time of diagnosis was 54.5 years. Five of them were women and 2 males. According to the Hans algorithm, 6 patients had the germinal centre B cells (GBC) type and one non-GBC (activated B-cell [ABC]) type, double expressor. Genetic profiling of four patients according to Schmitz's classification showed that 1 case was of the BN2 subtype, 1 of EZB subtype, 2 were unclassified. The six treated patients reached a complete response and did not experience progression of the disease during the median follow-up period of 80.5 months. CONCLUSIONS: We report on one of the largest series of CD56+DLBCL with detailed clinicopathological data and for the first time described genetical findings in a limited number of patients. Our results show that CD56 expression is rare, but seems to be present in prognostic favourable subtypes of DLBCL not otherwise specified (NOS) as tested by immunohistochemical or genetic profiling.

Cytopathological assessment is an accurate method for identifying immunophenotypic features and BRCA1/2 mutations of high-grade serous carcinoma from ascites.

BACKGROUND: High-grade serous carcinoma (HGSC) is the most common and aggressive type of ovarian cancer, and it is often associated with ascites at presentation. The objective of this study was to evaluate the accuracy of cytopathology to identify immunophenotypic features of HGSC and BRCA1/2 mutations from ascites. METHODS: The study included 45 patients with histologically confirmed primary HGSC and malignant ascites. Immunocytochemistry (ICC) staining for PAX8, WT1, P53, P16, and Ki-67 was performed on cytospins and cytoblocks prepared from ascites. Next-generation sequencing (NGS) was used to detect germline/somatic BRCA1/2 mutations in the ascites. Both ICC and NGS results were compared with immunohistochemistry (IHC) and NGS results from tissue blocks of primary tumor. Cronbach α and χ2 statistics, respectively, were used. RESULTS: ICC/IHC results for PAX8, WT1, P53, and P16 showed good reliability between cytospins, cytoblocks, and tissue blocks (α > 0.75), whereas poor reliability and significant differences were observed for Ki-67 between ascites and tissue blocks (α < 0.26; p < .001 [Kruskal-Wallis]). For germline BRCA1/2 mutations, 100% concordance was confirmed, but only 14% concordance was confirmed for somatic mutations. CONCLUSIONS: These results demonstrate that cytopathology is an accurate method for identifying immunophenotypic features of HGSC and detecting germline BRCA1/2 mutations from ascites. However, further investigation is required for assessing the proliferation activity of HGSC in ascites and for detecting somatic BRCA1/2 mutations.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

The impact of the COVID-19 pandemic on organised cervical cancer screening: The first results of the Slovenian cervical screening programme and registry.

BACKGROUND: The COVID-19 pandemic threatens the impact of cervical cancer screening and global cervical cancer elimination goals. As cervical cancer screening programmes were adjusting to the new situation, we evaluated the intensity, quality, and outcomes of cervical cancer screening in Slovenia in the first seven months of the pandemic. METHODS: Historical observational study on data from a population-based cervical cancer screening registry. Number of cervical cytopathology (screening and follow-up), histopathology (diagnostic procedures, invasive procedures and number of newly diagnosed CIN2+ cases) and HPV test results from the entire Slovenian women population between January 1st and September 30th 2020 were compared to a three-year average of the years 2017-19. FINDINGS: A two-month screening lock-down between March 12th and May 8th 2020 resulted in an epidemic deficit of screening (-92%), follow-up (-70%), and HPV triage tests (-68%), as well as invasive diagnostic (-47%) and treatment (-15%) of cervical lesions. Time to diagnosis and treatment did not increase; times to laboratory results fluctuated but stayed within standards. Slovenia has entered the second epidemic intending to add as little as possible to the pandemic deficit of screening smears (-23%) and yearly CIN2+ cases (-10%). Women aged 30-39 were most affected, with the highest pandemic deficit of screening smears (-26%) and yearly CIN2+ cases (-19%). INTERPRETATION: The pandemic has deeply affected all levels of our lives. New vulnerable groups and inequalities have emerged that require recognition and action. To prevent long-term increases in the cervical cancer burden due to the COVID-19 pandemic, it is crucial that organised screening is maintained and monitored in settings where it can be safely and comprehensively provided. FUNDING: None.

Morphological features of breast cancer circulating tumor cells in blood after physical and biological type of isolation.

BACKGROUND: Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Different isolation tech-niques based on their biological or physical features were established. Currently, the most widely used methods for visualization after their separation are based on immunofluorescent staining, which does not provide the information on the morphology. MATERIALS AND METHODS: The aim of this study was to evaluate how two different separation techniques affect cell morphology and to analyse cell morphology with techniques used in routine cytopathological laboratory. A direct side-by-side comparison of physical (Parsortix®) and biological (MACS®) separation technique was performed. RESULTS: In the preclinical setting, both isolation techniques retained the viability and antigenic characteristics of MCF7 breast cancer cells. Some signs of degeneration such as cell swelling, cytoplasmic blebs, villous projections and vacuolization were observed. In metastatic breast cancer patient cohort, morphological features of isolated CTCs were dependent on the separation technique. After physical separation, CTCs with preserved cell morphology were detected. After biological separation the majority of the isolated CTCs were so degenerated that their identity was difficult to confirm. CONCLUSIONS: Taken together, physical separation is a suitable technique for detection of CTCs with preserved cell morphology for the use in a routine cytopathological laboratory.

Potentiation of electrochemotherapy effectiveness by immunostimulation with IL-12 gene electrotransfer in mice is dependent on tumor immune status.

Electrochemotherapy (ECT) exhibits high therapeutic effectiveness in the clinic, achieving up to 80% local tumor control but without a systemic (abscopal) effect. Therefore, we designed a combination therapy consisting of ECT via intratumoral application of bleomycin, oxaliplatin or cisplatin with peritumoral gene electrotransfer of a plasmid encoding interleukin-12 (p. t. IL-12 GET). Our hypothesis was that p. t. IL-12 GET potentiates the effect of ECT on local and systemic levels and that the potentiation varies depending on tumor immune status. Therefore, the combination therapy was tested in three immunologically different murine tumor models. In poorly immunogenic B16F10 melanoma, IL-12 potentiated the antitumor effect of ECT with biologically equivalent low doses of cisplatin, oxaliplatin or bleomycin. The most pronounced potentiation was observed after ECT using cisplatin, resulting in a complete response rate of 38% and an abscopal effect. Compared to B16F10 melanoma, better responsiveness to ECT was observed in more immunogenic 4 T1 mammary carcinoma and CT26 colorectal carcinoma. In both models, p. t. IL-12 GET did not significantly improve the therapeutic outcome of ECT using any of the chemotherapeutic drugs. Collectively, the effectiveness of the combination therapy depends on tumor immune status. ECT was more effective in more immunogenic tumors, but GET exhibited greater contribution in less immunogenic tumors. Thus, the selection of the therapy, namely, either ECT alone or combination therapy with p. t. IL-12, should be predominantly based on tumor immune status.

Preclinical and Clinical Evaluation of Magnetic-Activated Cell Separation Technology for CTC Isolation in Breast Cancer.

Circulating tumor cell (CTC) count is an independent prognostic factor in early breast cancer. CTCs can be found in the blood of 20% of patients prior to neoadjuvant therapy. We aimed to assess the suitability of magnetic-activated cell separation (MACS) technology for isolation and cytological characterization of CTCs. In the preclinical part of the study, cell lines were spiked into buffy coat samples derived from healthy donors, and isolated using MACS. Breast cancer cells with preserved cell morphology were successfully isolated. In the clinical part, blood for CTC isolation was drawn from 44 patients with early and locally advanced breast cancer prior to neoadjuvant chemotherapy. Standard Giemsa, Papanicolaou and pancytokeratin staining was applied. 2.3% of samples contained cells that meet both the morphological and immunocytochemical criteria for CTC. In 32.6% of samples, partially degenerated pancytokeratin negative cells with morphological features of tumor cells were observed. In 65.1% of samples, CTCs were not found. In conclusion, our results demonstrate that morphologically intact tumor cells can be isolated using MACS technology. However, morphologically intact tumor cells were not detected in the clinical part of the study. At present, MACS technology does not appear suitable for use in a clinical cytopathology laboratory.

Chronic Lymphocytic Leukemia with Divergent Richter's Transformation into a Clonally Related Classical Hodgkin's and Plasmablastic Lymphoma: A Case Report.

Chronic lymphocytic leukemia (CLL) typically pursues a prolonged course. Its transformation into a more aggressive lymphoma occurs in 2-8% of all patients. Most commonly, diffuse large B-cell lymphoma develops. Transformation into a classical Hodgkin's lymphoma (cHL) occurs in <1%. Plasmablastic transformation has been only rarely reported. Cases of synchronous divergent transformation of CLL into a composite lymphoma are exceedingly rare. We describe the unique occurrence of the transformation of a long-standing CLL into a synchronous clonally related cHL as well as plasmablastic lymphoma (PBL) in an 85-year-old female patient. After 10 years of asymptomatic CLL, our patient was treated with a rituximab-chlorambucil scheme in combination with pegfilgrastim for recurrent infections and the development of B symptoms. Five cycles (of six planned) were administrated with no adverse effects. After the fifth cycle, lymphadenopathy with pronounced B symptoms appeared. Histology showed the presence of cHL in the lymph node, while the bone marrow was infiltrated by PBL. Our patient died in sepsis not receiving further specific oncologic treatment due to her poor general condition. Additional cytogenetic and molecular studies showed that this was a case of mutated CLL with trisomies of chromosomes 12, 3, and 18 (a rare specific +12 plus other-non+19 CLL subgroup). The presence of trisomy 12 has also been proved in plasmablasts and in cHL cells.

Usefulness of Bcl-2 Expression and the Expression of Cytoplasmic Immunoglobulin Light Chains in the Differentiation Between B-Cell Lymphoma and Reactive Lymphocytic Proliferations in FNA.

Flow cytometry is helpful in differentiating between B-cell lymphoma (BCL) and reactive lymphocytic proliferation (RLP) in FNA biopsies. However; the presence of inconclusive surface immunoglobulin light chains (sIg LC) poses a problem. We investigated the usefulness of additional tests; namely Bcl-2 expression and expression of cytoplasmic Ig LC (cIg LC), mainly on samples with inconclusive sIg LC. Both tests were performed on 232 FNA samples from lymph nodes. Bcl-2 alone was determined qualitatively and quantitatively on 315 samples. The quantitative test was correctly positive in 76% of cases and falsely negative in 24%. The correctly positive results of the qualitative test were 11% points lower. cIg LC correctly identified 65% of BCL with dual positive sIg LC; 36% of BCL with difficult to interpret sIg LC and only 7% of BCL with negative sIg LC. The best results in differentiating between BCL and RLP were obtained when all three tests were used together. In samples with inconclusive sIg LC and additional monoclonal or polyclonal populations the κ:λ ratios did not differentiate between RLP and BCL. We propose that in case of inconclusive sIg LC Bcl-2 test is used first. The addition of cIg LC test is sensible only in cases with dual positive and difficult to interpret sIg LC.

Interobserver variability and accuracy of p16/Ki-67 dual immunocytochemical staining on conventional cervical smears.

BACKGROUND: p16/Ki-67 dual immunocytochemical staining (DS) has been proven as a sensitive and specific test for triage of HPV positive women with good reproducibility and accuracy. However, implementation of the test into an organized screening program (OSP) is not easy. The aims of this study were to compare the performance and agreement of DS results among three Slovenian cytopathological laboratories involved in the national OSP, and to define cases where staining results can be difficult to interpret. METHODS: Cervical smears were obtained for DS from 129 women referred to colposcopy. Smears were evaluated blindly in three laboratories by a cytotechnologist and a cytopathologist after initial training. Results were positive, suspicious, negative or inadequate. Five characteristics of DS staining were recorded. After primary evaluation, an extensive expert-led additional training was undertaken, including a discussion of difficult cases and a practical exam. Smears were re-evaluated and results compared to primary evaluation. RESULTS: After the additional training, the overall percentage of agreement among the three laboratories increased from 77.5 to 89.9% and kappa increased from 0.70 to 0.86. Sensitivity for CIN2+ increased in two laboratories, to 90.5 and 85.7%, without the loss of specificity (75.8%). In one laboratory, the sensitivity slightly decreased from 90.5 to 88.9%, but the specificity increased from 63.6 to 68.2%. Difficult cases had significantly less DS cells, weak intensity of p16 staining, suboptimal cell morphology and background staining compared to positive cases. CONCLUSION: Additional expert-led training and discussion of difficult cases are necessary for accurate interpretation of DS in laboratories involved in OSP. The most difficult cases were those with single stained cells and weak p16 staining. Training protocol for safe implementation of p16/Ki-67 DS in OSP is proposed.

Bystander Effect Induced by Electroporation is Possibly Mediated by Microvesicles and Dependent on Pulse Amplitude, Repetition Frequency and Cell Type.

Bystander effect, a known phenomenon in radiation biology, where irradiated cells release signals which cause damage to nearby, unirradiated cells, has not been explored in electroporated cells yet. Therefore, our aim was to determine whether bystander effect is present in electroporated melanoma cells in vitro, by determining viability of non-electroporated cells exposed to medium from electroporated cells and by the release of microvesicles as potential indicators of the bystander effect. Here, we demonstrated that electroporation of cells induces bystander effect: Cells exposed to electric pulses mediated their damage to the non-electroporated cells, thus decreasing cell viability. We have shown that shedding microvesicles may be one of the ways used by the cells to mediate the death signals to the neighboring cells. The murine melanoma B16F1 cell line was found to be more electrosensitive and thus more prone to bystander effect than the canine melanoma CMeC-1 cell line. In B16F1 cell line, bystander effect was present above the level of electropermeabilization of the cells, with the threshold at 800 V/cm. Furthermore, with increasing electric field intensities and the number of pulses, the bystander effect also increased. In conclusion, electroporation can induce bystander effect which may be mediated by microvesicles, and depends on pulse amplitude, repetition frequency and cell type.

Inconclusive flow cytometric surface light chain results; can cytoplasmic light chains, Bcl-2 expression and PCR clonality analysis improve accuracy of cytological diagnoses in B-cell lymphomas?

BACKGROUND: Flow cytometric immunophenotyping (FCI), is widely used in cytology for distinguishing between B-cell lymphoma (BCL) and reactive lymphocytic proliferations (RLP), mainly by identifying monotypic B-cell populations. Since this cannot always be determined by ratios of surface immunoglobulin light chains (sIg LCs) we wanted to assess if cytoplasmic immunoglobulin (cIg) LCs, Bcl-2 and polymerase chain reaction (PCR) based clonality analysis can improve accuracy of cytological diagnoses of BCL. METHODS: Our study included 98 fine needle aspiration biopsies from lymph nodes suspicious for BCL with inconclusive sIg LCs. In all cases PCR clonality analysis was performed in order to determine immunoglobulin heavy chain (IGH) gene and T-cell receptor (TRC) gene rearrangement. In selected cases expression of Bcl-2 and cIg LC were determined by FC. RESULTS: Thirty patients had lymphoma and 68 had reactive lymphocytic proliferations. Three patterns of sIg LCs staining were found: negative, dual positive and difficult to interpret. Percentage of lymphomas was highest in the dual positive group (75 %). Morphology coupled with cIg LCs determination and/or Bcl-2 expression was able to give a correct diagnosis in 83 % of cases. Molecular tests would have been misleading in 15 % of cases because 7/30 BCL were polyclonal and 8/68 RLP were monoclonal. CONCLUSIONS: Determination of cIg LCs, Bcl-2 expression and PCR clonality analysis of B cells improved accuracy of cytological diagnoses in BCL with inconclusive sIg LC. However, clonality determined by PCR was misleading in some cases.

Hodgkin's lymphoma is a rare form of clonal haematological non-mast cell disease in systemic mastocytosis.

BACKGROUND: The association of systemic mastocytosis (SM) with a non-mast cell haematological neoplasm represents a specific subtype of mastocytosis termed systemic mastocytosis with associated haematological non-mast cell disease (SM-AHNMD). The overwhelming majority of the associated neoplasms are of myeloid origin, while lymphoid neoplasms associated with SM have been reported rarely. Association of SM with Hodgkin's lymphoma (HL) is exceedingly rare; so far, only two cases of HL as associated hematological non-mast cell disease in systemic mastocytosis have been published in the recent English literature. CASE: We present a case of a 37-year-old otherwise healthy male who was referred to our institution because of a one-month lasting dysphagia of both hard and liquid food. Physical examination showed tumour in the left jugular area measuring 2 cm in the largest diameter while computer tomography of the thorax revealed a 5.2 cm large, hypodense, soft tissue tumour between the trachea and left arteria carotis communis. On the basis of FNAB findings, the diagnosis of a "neutrophil-rich" Hodgkin's lymphoma was established. Excisional biopsy of mediastinal tumor showed lymphoid neoplasm with morphology and immunophenotype consistent with nodular sclerosis classical Hodgkin's lymphoma (NScHL). Bone marrow trephine biopsy and the MGG-stained smear of the bone marrow aspirate performed for lymphoma staging revealed an existence of systemic mastocytosis which was unexpected and incidental finding. Mast cells were highlighted by CD117 and tryptase immunostainings while CD25 positivity of mast cells was consistent with their neoplastic phenotype.There were no HL infiltrates present in the bone marrow. CONCLUSION: We report a very rare combination of systemic mastocytosis with Hodgkin's lymphoma as associated clonal haematological non-mast cell lineage disease. Systemic mastocytosis was an unexpected finding. The diagnosis of SM in bone marrow in our case was straight-forward, but it can be difficult in the case of reactive lymphoid aggregates or a difficult distinction between SM and HL infiltration. In particular, distinction can be challenging from the immunohistochemical point of view in the case of high-grade mast cell disease which can be CD30 positive.

Coexistent hairy cell leukaemia and hepatosplenic T-cell lymphoma: a case report.

BACKGROUND: Hairy cell leukaemia (HCL) is a chronic B-cell leukaemia characterized by expansion of neoplastic cells in the spleen, bone marrow and blood. Symptoms of HCL are related to pancytopenia and immune deficiency. Patients with HCL have an increased risk of second malignancy either in a form of synchronous disease or in a form of an increased incidence of a second neoplasm after the treatment of HCL. Hepatosplenic T-cell lymphoma (HSTCL) is a rare form of aggressive extranodal T-cell lymphoma. Its pathogenesis is connected to a chronic immune deficiency status and its coexistence with other neoplasms is practically non-existent. CASE: We present a case of a 53-year-old female patient suffering from hepatosplenomegaly, peripheral lymphadenopathy and related B symptoms. An excisional biopsy of the enlarged axillary lymph node revealed partial infiltration with CD3+/CD56+/TIA + T cell lymphoma. Bone marrow trephine biopsy and flow cytometric immunophenotypization of bone marrow cells and peripheral blood showed presence of two types of neoplastic cells in the peripheral blood and in the bone marrow (composite lymphoma). One of them showed typical morphologic characteristics and immunohistochemical features of HCL, while another one was morphologically and immunophenotypically consistent with the diagnosis of HSTCL, respectively. The patient was treated with multivalent chemotherapy including rituximab but all treatments turned out to be only partially effective. While HCL responded to the treatment, HSTCL was refractory to the chemotherapy and the patient died 7 months after the initial diagnosis because of haematemesis induced by Mallory-Weiss syndrome. CONCLUSION: This is the first recorded case of coexistent HCL and HSTCL in the same patient. A multidisciplinary approach, encompassing careful morphology interpretation, immunophenotypic, cytogenetic and molecular analyses, is mandatory to obtain an accurate diagnosis of composite lymphoma. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9354870531161685.

A flow cytometric analysis of vitreous inflammatory cells in patients with proliferative diabetic retinopathy.

The purpose of this study was to investigate inflammatory cells in vitreous from patients with proliferative diabetic retinopathy (PDR) using flow cytometric analysis. Twenty-eight patients with PDR requiring vitrectomy because of macular traction or tractional retinal detachment were enrolled in the study (n = 28), and 6 patients with macular hole (MH) formed the control group. Samples of vitreous and peripheral venous blood were obtained at the beginning of vitrectomy. T lymphocytes were found in vitreous from patients with PDR, and CD4/CD8 ratio was higher in vitreous (median 4.3) compared to blood (median 1.9; P = 0.003). No B lymphocytes were detected in vitreous. The percentage of histiocytes/macrophages was significantly higher in vitreous (median 62.1) in comparison with blood (median 5.5; P < 0.0001). No lymphocytes were detected in vitreous of the control group. There were more T lymphocytes in vitreous from patients with active PDR. No association between cells in the vitreous and visual acuity improvement after surgery was found. In conclusion, T lymphocytes are found in vitreous from patients with PDR and reflect the activity of PDR but do not seem to predict visual prognosis. Higher CD4/CD8 ratio in vitreous compared to blood from patients with PDR is consistent with local inflammatory response in PDR.