T-cell phenotypes in bronchoalveolar lavage fluid, blood and lymph nodes in pulmonary sarcoidosis--indication for an airborne antigen as the triggering factor in sarcoidosis.

BACKGROUND: An increased percentage of CD4+ T cells is usually observed in bronchoalveolar lavage fluid (BALF) from patients with sarcoidosis. In HLA-DRB1*03-positive patients, such T cells express the T-cell receptor (TCR) AV2S3+ gene segment. It is not known whether cells found in BALF reflect those in enlarged regional lymph nodes (LNs). Therefore, the aim of this study was to compare T-cell phenotypes in BALF, blood and mediastinal LNs. METHODS: Fifteen patients underwent clinical investigation including bronchoscopy with bronchoalveolar lavage. Blood samples were drawn, and endoscopic ultrasound-guided fine-needle aspiration of enlarged mediastinal LNs was performed via the oesophagus. T cells from all three compartments were analysed by flow cytometry for markers of activity, differentiation and T regulatory function. RESULTS: The CD4/CD8 ratio was significantly higher in BALF compared with regional LNs and was also significantly higher in LNs than in blood. The CD4+ T cells were recently activated and more differentiated in BALF than in blood and LNs. There was an accumulation of T regulatory cells (FOXP3+) in LNs and a correlation between high levels of FOXP3+ cells in BALF and in LNs. In HLA-DRB1*03-positive patients, TCR AV2S3+ CD4+ T cells were predominantly localized within BALF. CONCLUSIONS: The CD4+ T-cell phenotype in BALF indicates an active ongoing specific immune response primarily localized to the alveolar space.

EAHP 2020 workshop proceedings, pediatric myeloid neoplasms.

The first section of the bone marrow workshop of the European Association of Haematopathology (EAHP) 2020 Virtual Meeting was dedicated to pediatric myeloid neoplasms. The section covered the whole spectrum of myeloid neoplasms, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). The workshop cases are hereby presented, preceded by an introduction on these overall rare diseases in this age group. Very rare entities such as primary myelofibrosis, pediatric MDS with fibrosis, and MDS/MPN with JMML-like features and t(4;17)(q12;q21); FIP1L1::RARA fusion, are described in more detail.

Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting.

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 5.2 × 10-3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10-4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

Inflammation and tissue repair markers distinguish the nodular sclerosis and mixed cellularity subtypes of classical Hodgkin's lymphoma.

BACKGROUND: Classical Hodgkin's lymphoma (cHL), although a malignant disease, has many features in common with an inflammatory condition. The aim of this study was to establish the molecular characteristics of the two most common cHL subtypes, nodular sclerosis (NS) and mixed cellularity (MC), based on molecular profiling and immunohistochemistry, with special reference to the inflammatory microenvironment. METHODS: We analysed 44 gene expression profiles of cHL whole tumour tissues, 25 cases of NS and 19 cases of MC, using Affymetrix chip technology and immunohistochemistry. RESULTS: In the NS subtype, 152 genes showed a significantly higher expression, including genes involved in extracellular matrix (ECM) remodelling and ECM deposition similar to wound healing. Among these were SPARC, CTSK and COLI. Immunohistochemistry revealed that the NS-related genes were mainly expressed by macrophages and fibroblasts. Fifty-three genes had a higher expression in the MC subtype, including several inflammation-related genes, such as C1Qalpha, C1Qbeta and CXCL9. In MC tissues, the C1Q subunits were mainly expressed by infiltrating macrophages. CONCLUSIONS AND INTERPRETATIONS: We suggest that the identified subtype-specific genes could reflect different phases of wound healing. Our study underlines the potential function of infiltrating macrophages in shaping the cHL tumour microenvironment.

Ten-color 15-antibody flow cytometry panel for immunophenotyping of lymphocyte population.

We have developed a lymphoproliferative disorder screening tube (LPD-ST) with the aim to provide comprehensive immunophenotyping of lymphocyte subsets with minimal need for additional testing. The LPD-ST consists of CD4/kappa FITC, CD8/lambda PE, CD3/CD14ECD, CD38PC5.5, CD20/CD56PC7, CD10APC, CD19APC-A700, CD5APC-A750, CD57/CD23PB and CD45KO. The LPD-ST was validated against previously used lymphocyte subset panels in Canada (n=60) and in Sweden (n=43) and against the OneFlow™ LST (n=60). The LPD-ST panel was then implemented in clinical practice using dried monoclonal antibody reagents (Duraclone® ) on 649 patient samples in Sweden. In 204 of 649 samples (31%), a monotypic B-cell population was found. Of these cases, a final diagnosis could be rendered in 106 cases (52%), and in the remainder, additional B-cell immunophenotyping was performed. In 20 (3%) samples, an aberrant T-cell population was confirmed by additional testing. Of 425 samples diagnosed as normal/reactive lymphoid tissue, 50 (12%) required additional immunophenotyping, mostly due to an abnormal CD4/CD8 ratio. The LPD-ST tube significantly minimizes the need for additional testing, improves the turn-around time, and reduces the cost of LPD immunophenotyping. It is also suitable for investigating paucicellular samples such as cerebrospinal fluid or fine needle aspirates.

Acute lymphoblastic leukemias from relapse engraft more rapidly in SCID mice.

It is generally believed that relapse of acute leukemia heralds progression of the disease into a more aggressive stage. The biological behavior of leukemic cells collected from four patients with adult acute lymphoblastic leukemia (ALL) prior to treatment and at relapse was studied after engraftment into 28 unconditioned mice with severe combined immunodeficiency (SCID). Leukemic cells engrafted in all but one mouse, with major differences observed in the growth and aggressiveness of the leukemias. Recipient mice of cells derived from all patients at relapse died more rapidly in overt leukemia than those which were injected with cells obtained prior to induction treatment (P=0.0002). SCID mice that received cells from one patient at the time of diagnosis also died in terminal leukemia. Other SCID mice however, that received cells from the remaining three patients prior to treatment developed occult leukemia that was detectable in the blood or bone marrow with the use of polymerase chain reaction (PCR) or flow cytometry only. Leukemic cells recovered from mice with terminal leukemia exhibited a larger proliferating fraction than cells originally injected (P=0.004). Our results demonstrate, that during the evolution from initial presentation to relapse, ALL cells may acquire biological properties which render them more aggressive in SCID mice.

Flow cytometry immunophenotyping in integrated diagnostics of patients with newly diagnosed cytopenia: one tube 10-color 14-antibody screening panel and 3-tube extensive panel for detection of MDS-related features.

Acute leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms and lymphomas are the most prevalent diagnoses in adults presenting with new onset cytopenia. Here, we describe two 10-color panels of surface markers (screening and comprehensive panel) applied at the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada. A 10-color flow cytometry is applied using the stain-lyse-wash sample preparation method. In patients with <10% blasts and no clear involvement by hematological malignancy based on cytomorphological evaluation of bone marrow (BM) smear, the recently published one-tube 10-color 14-antibody screening panel is applied. This panel allows detection of major B- and T-cell abnormalities, enumeration of cells in blast region (CD45 dim), and gives insight into myeloid BM compartment, including calculation of four-parameter score for MDS-related abnormalities. In patients who present with ≥10 - <20% blasts in blood or BM smears, a comprehensive three-tube panel of surface markers is used up front. The analysis is focused on the detection of abnormal antigen expression patterns not seen in normal/reactive BM, according to the guidelines developed by International/European LeukemiaNet Working Group for Flow Cytometry in MDS. In patients with ≥20% blasts, an additional tube is added to allow the detection of cytoplasmic markers necessary to diagnose mixed phenotype acute leukemia.

ICSH guidelines for the standardization of bone marrow immunohistochemistry.

Bone marrow (BM) tissue biopsy evaluation, including trephine biopsy and clot section, is an integral part of BM investigation and is often followed by ancillary studies, in particular immunohistochemistry (IHC). IHC provides in situ coupling of morphological assessment and immunophenotype. The number of different IHC tests that can be applied to BM trephine biopsies and the number of indications for IHC testing is increasing concurrently with the development of flow cytometry and molecular diagnostic methods. An international Working Party for the Standardization of Bone Marrow IHC was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines for the standardization of BM IHC based on currently available published evidence and modern understanding of quality assurance principles as applied to IHC in general. The guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus and represent further development of the previously published ICSH guidelines for the standardization of BM specimens handling and reports.

DNA content and expression of PCNA and p53 in Hodgkin's disease and Hodgkin's-like B-cell lymphoma.

DNA ploidy (by image cytometry) and expression of proliferating cell nuclear antigen (PCNA) and p53 tumor suppressor gene product (by immunohistochemistry) were investigated in 15 cases of Hodgkin's disease (HD) and 12 cases of HD-like B-cell lymphoma (HD-like NHL). Reed-Sternberg (RS) cells and their variants were DNA aneuploid in all cases. However, the fraction of hyperoctaploid tumor cells was higher in HD than in HD-like NHL. PCNA expression was high in neoplastic cells (> 50%) and variable (5-40%) in reactive lymphocytes in both HD and HD-like NHL. p53 positivity was found in RS cells and their variants in 64% of HD cases, but only in 25% of cases of HD-like NHL. Our results support the suggestion that HD-like B-cell lymphomas should be considered as highly malignant non-Hodgkin's lymphomas rather than Hodgkin's disease.

Follicular involution in HIV lymphadenopathy. A morphometric study.

Lymph node biopsies from 75 HIV infected patients (71 homo- and bisexual men, 3 hemophiliacs and 1 woman) were studied using immunohistochemical methods with monoclonal antibodies (Mabs) against B lymphocytes, subsets of T lymphocytes, follicular dendritic cells (FDC) and HIV gag proteins p24 and p18. Histopathological changes were classified as follicular hyperplasia (FH), fragmentation (FF), atrophy (FA) and depletion (FD). Immunohistochemical stainings were quantified with the help of an Image Quantifier (IQ) and the reactivity for respective Mab-defined antigen was related quantitatively to other antigens and histopathological changes. Such measurements showed an increase in FDC in biopsies with FH and FF histology and a decrease in FA and FD cases in comparison with cases with non-HIV related lymphadenopathy. In addition it was found that the decrease in FDC was correlated with an increase in CD8+ within the follicles. Double immunostainings for p24 and various cellular markers showed that p24 was predominantly associated with follicular dendritic cells. Essentially the same findings were observed in the lymph nodes irrespective of risk group. Possible mechanisms involved in follicular involution in HIV-related lymphadenopathy are discussed.

Cell association of HIV in AIDS-related encephalopathy and dementia.

The presence of HIV gag and env proteins (HIV Ag) and virus replicating cells was studied by immunohistochemistry and in situ hybridization, respectively, in brain specimens from five HIV infected patients. HIV antigens were detected in 3 of 5 brains in micronodular areas characterized by increased cellularity and the presence of multinuclear giant cells. By double immunostaining, HIV Ag positive cells were shown to express markers common to macrophages and microglia i.e. Leu M5+, My4+, HLA-Dr+, RCA-1+, and to a lesser extent CD4+ (Leu3+). Another macrophage specific marker, KiM6, was found only on HIV+ cells in HIV infected specimens and not in uninfected, control brains. Medium-sized, virus replicating cells were found exclusively in micronodular areas, but in much smaller quantities than HIV Ag+ cells. Our observations provide further evidence to support the hypothesis that macrophages play an important role in CNS infection by HIV and additionally support the concept that reactive microglial originate from activated macrophages infiltrating the brain. Both direct effects of viral components and cell mediated reactions can be implicated from our findings as mechanisms involved in the pathogenesis of the CNS lesions.

Method to quantitate intestinal metaplasia of stomach by image analysis.

With the aid of an image quantifier, the distribution of histochemically labelled (alcian blue, pH 2.5) mucin-producing goblet cells was recorded from a gastrectomy specimen with a peptic ulcer to determine the degree of metaplasia. Of 254 measurements made, 130 were in the antrum and 124 in the fundic area. The areas occupied by cells positive for alcian blue were 7.5 (SD 9.89)% in the antral region, and 1.8 (1.84)% and 0.92 (1.15)% in the two zones representing the fundic area. The difference between the positive mucosal areas in the antrum was significantly higher (p less than 0.001) than in the fundic area. The positively stained area found along the lesser curvature was 7.76 (12.0)% while along the greater curvature, it was 2.17 (3.02)% (p less than 0.001). This method will be useful for future studies of the extent and topographical distribution of intestinal metaplasia among populations with disparate incidences of gastric carcinoma because it permits comparison of different areas of intestinal metaplasia in mucous gastric zones.

Expression of P-Glycoprotein in Non-Hodgkin's Lymphomas.

Drug resistance has been shown to be associated with the expression of P-glycoprotein (P-gp), the product of the mdr-1 gene. In the present study the expression of P-gp in 57 cases B-cell non Hodgkin lymphoma NHL was assessed before chemotherapy. Six cases of reactive lymphoid tissue and 11 cases of solid tumors were also studied. The expression of P-gp was evaluated by immunocyto- and histochemical methods, using three different Monoclonal Antibodies C219, JSB-1 and MRK16 directed against separate epitopes of P-gp. Comparable frequencies of cases positive for P-gp were found in low grade (6/40) and high grade (3/17) lymphomas. The pattern of staining was predominantly cytoplasmic, although a Golgi-associated dot like pattern of staining was also seen, mostly with JSB-1 MAb. Both cases of Hairy cell leukemia were P-gp positive. P-gp expression was also found in the endothelium of small capillaries and some high endothelial venules, as well as in macrophages, in both lymphomas and reactive lymphoid tissues. P-gp expression was found in a low frequency in NHL, suggesting that clinical drug resistance may already be predicted at the time of diagnosis and thus may serve as a guide in the choice of chemotherapeutical regiment.

Intermittent infusion of cladribine (CdA) in previously treated patients with low-grade non-Hodgkin's lymphoma.

Thirty-six patients with previously treated low-grade non-Hodgkin's lymphoma (LG-NHL) were included in a phase II study between August 1990 and February 1994 and treated with 0.12 mg/kg CdA as a 2 h.i.v. infusion daily x V, q 28 days up to 6 courses. Twenty-three were refractory to previous chemotherapy while 13 were relapsed. Four patients had mantle cell lymphoma, 17 follicle centre cell derived lymphoma, 7 lymphoplasmacytoid lymphomas and, 8 had small lymphocytic lymphoma. The response rate was 42%, with 5 (14%) CR and 10 (28%) PR while 6 (16%) patients progressed during treatment. The median number of delivered CdA courses was 3 (1-6) in non-responding cases and 6 (2-6) in responders. The median time to progression was 9 mo for all patients, 23 mo for CR and 16 mo for PR patients. Toxicity was sometimes severe with 3 infectious deaths (1 pneumocystis carinii pneumonia, 1 gram negative septicemia, and 1 fungal pneumonia), and 6 grade 3 or 4 infectious episodes. We conclude that responses to CdA in this group of heavily pre-treated patients is impressive. However, toxicity is considerable and the rate of opportunistic infections is worrisome.

Effect of Cytotoxic Cells on Minimal Residual Leukemia.

The presence of minimal residual disease is indicated by the high frequency of relapses after twin bone marrow transplants and after allogeneic bone marrow transplants without graft versus host disease (up to 75% and 45% of cases, respectively). The graft versus leukemia effect may be mediated by IL-2 activation of natural killer cells (CD16 +, CD56 +, CD3-, CD8+/-) or cytotoxic T cells (CD3 +, CD56+/-). These activated killer cells can bind to targets and cause their lysis, and then recirculate to kill other targets. Killing can be blocked by anti-perforin antibodies and enhanced by protein kinase C-activation of effectors There are several studies indicating that a high percentage of leukemic cells can be killed by LAK-cells. However even in the most sensitive cases, lysis of all the cells cannot be achieved. The finding that leukemic clonogenic cells are generally sensitive to both NK and LAK cytotoxicity provides a more hopeful possibility The lack of tumor specific antigents, leukemic cell-immunoheterogeneity and maturation asynchrony explains why antigen dependent T-cell mediated cytotoxicity is only partly effective in eradicating residual leukemic cells. Future work should therefore include more studies of the mechanism of resistance to LAK cells, possibilities of further enchancing cytotoxicity and the mechanism of graft-versus-leukemia effect.

Conjugate formation by leukemic blasts from acute myeloid leukemia with cytotoxic lymphocytes.

Conjugate formation by AML blasts with fresh peripheral blood lymphocytes (PBL) and lymphokine activated killer (LAK) effectors was studied by flow cytometry. Leukemic blasts formed very low numbers of conjugates with fresh PBL and were resistant to natural killer (NK) cytotoxicity. When LAK effectors were used a significant increase in conjugate formation was observed, which in the majority of cases was followed by an increased killing. There was a positive correlation between the percentages of conjugates formed by AML blasts with LAK effectors and the susceptibility to lysis. No significant difference in binding activity between the CD3+ and CD56+ LAK subpopulations was found. There was no correlation between the expression of ICAM-1, LFA-3 and Transferrin receptor (CD71) and the conjugate formation. The blocking of CD71 on the control K562 cell line reduced the conjugate formation with LAK effectors but no such effect could be observed with CD71+ AML blasts.

DNA image analysis in childhood acute lymphoblastic leukemia.

DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid--8 hyperdiploid and 1 hypodiploid. The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.

Cladribine for untreated or early low-grade non-Hodgkin's lymphoma.

PATIENTS AND METHODS: Forty-four patients, with low-grade non-Hodgkin's lymphoma (LG-NHL) were included in a phase II study between June 1993 and May 1995 and treated with cladribine (CdA) 0.12 mg/kg as a 2 h i.v. infusion daily x 5, repeated after 28 days for up to 6 courses. Thirty-four patients were previously untreated and 10 had progressive disease after initial response to limited chlorambucil treatment. Five patients had also received involved field radiotherapy. Eight patients had mantle cell lymphomas, 22 follicle centre lymphomas, 5 lymphoplasmacytoid lymphomas, 4 small cell lymphocytic lymphomas, 4 marginal zone B-cell lymphomas and I had unclassified low-grade NHL. The response rate was 64%, with 11 (25%) CR and 17 (39%) PR while 5 (11%) patients progressed during treatment. The response rate was similar in previously treated and untreated patients. The median number of CdA courses delivered was 3 (1-6) in non-responding patients and 6 (2-6) in responders. Median survival from inclusion was not reached with a median follow-up of 40 months. The median time to progression was 7 mo for all patients, 25+ mo for CR and 16 mo for PR patients. Toxicity was sometimes severe with 2 treatment related deaths, one infectious related and one due to a mucocutaneous syndrome and pulmonary microembolism. In addition, 5 grade 3 or 4 infectious episodes were seen. Seven patients experienced grade 3 or 4 thrombocytopenia and 20 had grade 3 or 4 neutropenia. We conclude that the majority of patients with low-grade non-Hodgkin's lymphoma respond to CdA but that the adverse effects may be severe.

IFN-gamma is not the only mediator of suppressed myelopoiesis produced by mononuclear cells from aplastic anemia patients.

Peripheral blood mononuclear cells (PBMC) from patients with aplastic anemia (AA) and healthy donors were compared with regard to their ability to produce soluble factors with inhibitory activity on in vitro granulopoiesis (GM-CFC). Although PBMC from AA patients produced enhanced levels of IFN-gamma as compared to controls, this lymphokine was found not to be the main inhibitor of in vitro granulopoiesis. Other, non-IFN related factors were potent inhibitors of both the mature and the immature precursors for GM-CFC, could act across the species barrier and were of low molecular weight. Also PBMC from healthy donors produced a non-IFN mediated GM-CFC inhibitory factor, but to a lesser degree and acting only on one type of myeloid precursors. The possible implications of these findings in relation to the etiology of AA will be discussed.

Immunocytochemical analysis and cytomorphologic diagnosis on fine needle aspirates of lymphoproliferative disease.

Fine needle aspirates were used for the cytologic and immunologic analysis of 21 cases of lymphoproliferative disorders. Immunocytochemical studies performed on Cytospin preparations confirmed the cytomorphologic diagnosis in 19 cases. In one case, the morphology of both aspirates and surgically obtained material showed a reactive pattern while immunologic analyses were inconclusive on both types of material. Immunocytochemistry on tumor material obtained by fine needle aspirations was in agreement with immunohistochemistry on surgical biopsies in 15 of 16 patients with malignant lymphomas. We conclude that immunocytochemical studies performed on Cytospin material in conjunction with the cytologic diagnosis will lead to an increase in diagnostic accuracy as well as providing a means for subclassification of neoplastic lymphoid cells. Moreover, this technique appears to give results comparable to those obtained by histopathologic and immunohistochemical analysis on surgically removed lymph nodes.