Molecular subtyping of gastric cancer according to ACRG using immunohistochemistry - Correlation with clinical parameters.

BACKGROUND: Gastric cancer (GC) is a very heterogenous disease necessitating further stratification for prognostic and therapeutic aspects. Based on the recommendation of The Asisan Cancer Research Group (ACRG) recently established four molecular subtypes (MSI, MSS/EMT, MSS/TP53+, MSS/TP53-) which require molecular expression analysis. The technology required for comprehensive molecular analysis is expensive and not applicable for routine diagnostics. Thus, in this study we established a classification system utilizing immunohistochemistry and morphology-based analyses as surrogate markers in order to reproduce the ACRG molecular subtypes of gastric cancer. To clarify the clinical relevance of the novel classification system, we performed a correlation with established clinical parameters. METHODS: The study cohort consisted of 189 patients with GC (UICC III and IV). Using immunohistochemistry, the following markers were analysed: MLH1, MSH2, MSH6, PMS2 (as a surrogate for microsatellite status), p53, SOX9. We assessed tumor budding as a surrogate for EMT to distinguish between MSS/EMT and MSS/non-EMT groups. RESULTS: Immunohistochemical and morphologic subtyping classified cases as follows: 10% MSI, 35% MSS/EMT, 16% MSS/TP53 + and 39% MSS/TP53-. Subtypes significantly correlated with the Lauren classification, tumor stage, venous invasion and SOX9 expression (p < .05). There was no significant correlation between molecular subtype and lymph node growth pattern. CONCLUSION: We propose a simple algorithm for molecular subtyping of GC using universally available immunohistochemistry, which correlates with clinical parameters and is cost-effective and applicable in diagnostic routine. This classification might prospectively help to determine patient prognosis, optimize patient care and homogenize patient cohorts for clinical trials.

Deep phenotyping of T cell populations under long-term treatment of tacrolimus and rapamycin in patients receiving renal transplantations by mass cytometry.

Tacrolimus (FK506) and rapamycin (RAPA) are widely used to maintain long-term immunosuppression after organ transplantation. However, the impact of accumulative drug administration on the recipients' immune systems remains unclear. We investigated the impact of 3-year FK506 or RAPA treatment after renal transplantation on the human immune systems. A discovery cohort of 30 patients was first recruited, and we discovered two distinctive T lineage suppressive regulatory patterns induced by chronic treatment of FK506 and RAPA. The increased percentage of senescent CD8+ CD57+ T lineages and less responsive T cell receptor (TCR) pathway in the FK506 group indicate better graft acceptance. Meanwhile, percentages of regulatory T cells (Tregs) and expression of CTLA-4 were both up to two-fold higher in the RAPA group, suggesting the inconsistent reactivation potential of the FK506 and RAPA groups when an anti-tumour or anti-infection immune response is concerned. Additionally, up-regulation of phosphorylated signaling proteins in T lineages after in vitro CD3/CD28 stimulation suggested more sensitive TCR-signaling pathways reserved in the RAPA group. An independent validation cohort of 100 renal transplantation patients was further investigated for the hypothesis that long-term RAPA administration mitigates the development of tumours and infections during long-term intake of immunosuppressants. Our results indicate that RAPA administration indeed results in less clinical oncogenesis and infection. The deep phenotyping of T-cell lineages, as educated by the long-term treatment of different immunosuppressants, provides new evidence for personalized precision medicine after renal transplantations.

Clinical Aspects, Immunophenotypic Analysis and Survival Rate of Chronic Lymphocytic Leukaemia Patients in Erbil City, Iraq.

OBJECTIVES: Chronic lymphocytic leukaemia (CLL) is characterised by an accumulation of clonal B cells in the blood, bone marrow and lymphatic tissue. This study aimed to evaluate the clinical and immunophenotypic characteristics and survival rate of CLL patients. METHODS: This retrospective study was conducted at the Nanakaly Hospital for Blood Diseases & Oncology in Erbil, Iraq, between January 2011 and December 2017. A total of 105 CLL patients were assessed to determine clinical presentation and staging, immunophenotype and survival rate. RESULTS: The median age of the patients was 65 years and 63.8% were male. The main clinical presentations were splenomegaly (64.8%), pallor (61.9%) and lymphadenopathy (60%). More than half of the patients presented at an advanced clinical stage according to the Rai and Binet staging systems (59.1% and 55.2%, respectively). All CLL cases expressed both cluster of differentiation (CD)19 and CD5, 67.6% had monoclonal kappa light chains and 21% expressed CD38. The five-year overall survival (OS) rate was 61.3%. The mean duration of five-year survival was 41.3 months (95% confidence interval: 36.4-46.3 months). There were no correlations between survival and sociodemographic, clinical or laboratory characteristics. CONCLUSION: In comparison to the existing Western literature, Iraqi CLL patients more frequently presented with hepatosplenomegaly and at a more advanced clinical stage. In addition, the five-year OS rate was much lower.

ACUTE LEUKEMIAS IMMUNOPHENOTYPES AT AGAKHAN UNIVERSITY HOSPITAL, NAIROBI.

OBJECTIVE: The aim was to determine relative frequencies of acute leukemia immunophenotypes using commonly expressed markers and to describe the clinicopathological characteristics. Design: This was a prospective cross-sectional study. SETTING: The study was based at Aga khan clinical laboratory department. SUBJECTS: One hundred and thirty two (132) consecutive blood and bone marrow specimens from patients suspected to have acute leukemia were analysed for cytomorphological characteristics and immunophenotyping. The clinical-pathological characteristics were also recorded. Immunological category was assigned using the EGIL criteria. RESULTS: There were 88 AML and 42 ALL patients analysed for immunophenotypes. Only tw cases of biphenotypic leukemia were found. The commonest overall AML morphological sub-type was AML-M2, 26 (29.5%). Majority of ALL cases were B-cell immunological sub-type (96.6%). Early pre-B phenotype constituted 62.07% and Common B-cell ALL 37.93%. There were only 4 cases of T-cell ALL. Majority of patients presented with anaemia with a median hemoglobin of 7.5g/dl (range 2-15g/dl). The median platelet count was 55 (range 4-462 x 10(9)/L). CONCLUSION: Immunophenotyping of acute leukemia is beneficial in accurate diagnosis of patients with these malignancies in this setup. T-cell ALL, AML-M6 and M7 are less frequent than what has been reported in most studies in Africa.

Mixture modeling approach to flow cytometry data.

Flow Cytometry has become a mainstay technique for measuring fluorescent and physical attributes of single cells in a suspended mixture. These data are reduced during analysis using a manual or semiautomated process of gating. Despite the need to gate data for traditional analyses, it is well recognized that analyst-to-analyst variability can impact the dataset. Moreover, cells of interest can be inadvertently excluded from the gate, and relationships between collected variables may go unappreciated because they were not included in the original analysis plan. A multivariate non-gating technique was developed and implemented that accomplished the same goal as traditional gating while eliminating many weaknesses. The procedure was validated against traditional gating for analysis of circulating B cells in normal donors (n = 20) and persons with Systemic Lupus Erythematosus (n = 42). The method recapitulated relationships in the dataset while providing for an automated and objective assessment of the data. Flow cytometry analyses are amenable to automated analytical techniques that are not predicated on discrete operator-generated gates. Such alternative approaches can remove subjectivity in data analysis, improve efficiency and may ultimately enable construction of large bioinformatics data systems for more sophisticated approaches to hypothesis testing.

A new automated flow cytometry data analysis approach for the diagnostic screening of neoplastic B-cell disorders in peripheral blood samples with absolute lymphocytosis.

Currently, multiparameter flow cytometry immunophenotyping is the selected method for the differential diagnostic screening between reactive lymphocytosis and neoplastic B-cell chronic lymphoproliferative disorders (B-CLPD). Despite this, current multiparameter flow cytometry data analysis approaches still remain subjective due to the need of experienced personnel for both data analysis and interpretation of the results. In this study, we describe and validate a new automated method based on vector quantization algorithms to analyze multiparameter flow cytometry immunophenotyping data in a series of 307 peripheral blood (PB) samples. Our results show that the automated method of analysis proposed compares well with currently used manual approach and significantly improves semiautomated approaches and, that by using it, a highly efficient discrimination with 100% specificity and 100% sensitivity can be made between normal/reactive PB samples and cases with B-CLPD based on the total B-cell number and/or the sIgkappa+/sIglambda+ B-cell ratio. In addition, the method proved to be able to detect the presence of pathologic neoplastic B-cells even when these are present at low frequencies (<5% of all lymphocytes in the sample) and in poor-quality samples enriched in 'noise' events.

A scoring system based on the expression of six surface molecules allows the identification of three prognostic risk groups in B-cell chronic lymphocytic leukemia.

We have previously identified 12 surface antigens whose differential expression represented the signature of B-cell chronic lymphocytic leukemia (B-CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B-CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B-CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log-rank statistics were chosen to define the optimal cut-off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut-off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B-CLL patients, into three distinctive prognostic groups (P = 4.78 x 10(-11)) with high- (score 0-3), intermediate- (score 4-6), and low- (score 7-9) risk of death. The three risk groups showed different distribution of cases as for Rai's stages, IgVH mutations, and ZAP-70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B-CLL.

Immunophenotypic analysis of peripheral blood stem cell harvests from patients with multiple myeloma.

BACKGROUND AND OBJECTIVES: Four-color multiparameter immunophenotyping has recently proven to be an attractive technique for evaluating the plasma cell (PC) compartment since it allows discrimination between myelomatous and normal PC. This study was designed to investigate: i) whether peripheral blood is less contaminated than bone marrow as a source for an autologous transplant; ii) the effect of growth factors on mobilizing myelomatous PC into peripheral blood; iii) the degree of contamination by myelomatous PC in apheresis samples; and iv) whether the number of PC increases during the last days of apheresis. DESIGN AND METHODS: Using 4-color antigen staining we investigated the composition of the PC compartment in 90 apheresis products from 40 patients with MM; in 17 cases bone marrow and peripheral blood samples were also simultaneously evaluated. RESULTS: (i) All pre-mobilization bone marrow samples analyzed were always contaminated with myelomatous PC whereas only 41% of the post-mobilization peripheral blood samples were contaminated. Moreover, the use of peripheral blood would lead to a reduction of >5x10(5) infused myelomatous PC; (ii) mobilization with cytokines increased the number of circulating PC, generally because of an expansion of the normal PC population; (iii) forty-eight percent of all peripheral blood stem cell harvests were contaminated with myelomatous PC, although normal PC usually represented the predominant population; (iv) no significant changes were observed in the amount of contaminating myelomatous PC during the first three days of apheresis. INTERPRETATION AND CONCLUSIONS: Multiparameter immunophenotyping is a useful approach for investigating the PC compartment in apheresis products.

Genetic alterations in primary cutaneous CD30+ anaplastic large cell lymphoma.

Primary cutaneous CD30+ anaplastic large cell lymphoma (C-ALCL) represents a distinct clinical subtype of CD30+ anaplastic large cell lymphomas. The etiology and underlying molecular pathogenesis of C-ALCL remain unclear. This study aimed to investigate genetic changes in C-ALCL. Comparative genomic hybridization (CGH) analysis of 23 DNA samples from 15 C-ALCL cases identified chromosome imbalances (CI) in 10 samples from eight cases (43%). The mean number of CI per sample was 2.09 +/- 3.86, with gains (2.00 +/- 3.85) more common than losses (0.09 +/- 0.29). The most frequent CI were gains of 1/1p and 5 (50%) and 6, 7, 8/8p, and 19 (38%). Microarray-based CGH analysis of six DNA samples from five cases with CI revealed genomic imbalances (GI) in all of the cases studied. This included oncogene copy number gains of FGFR1 (8p11) in three cases, and NRAS (1p13.2), MYCN (2p24.1), RAF1 (3p25), CTSB (8p22), FES (15q26.1), and CBFA2 (21q22.3) in two cases. Real-time PCR analysis of nine DNA samples from eight cases with CI and GI detected amplifications of CTSB and RAF1 in seven cases (88%), REL (2p13p12) and JUNB (19p13.2) in six cases (75%), and MYCN and YES1 (18p11.3) in four cases (50%). Immunohistochemical staining of paraffin sections from six cases demonstrated expression of JUNB protein in five cases and BCL2 in three cases. These results reveal a consistent pattern of genetic alterations in C-ALCL and provide the molecular basis for further investigation of this disease.

Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients.

Ten years ago, we made an incidental flow cytometric observation while immunophenotyping biopsy and marrow samples from children suspected to have leukemia/non-Hodgkin's lymphoma, but were subsequently diagnosed with neuroblastoma. The samples contained neoplastic CD45(-) cells that had an extremely bright CD56(+) (beyond the fourth decade on a four-decade scale) population distinguishable from CD45(+)CD56(usual density+) natural killer lymphocytes as well as other CD45(-)CD56(usual density+) nonhematopoietic tumors such as small cell carcinoma or melanoma. Following the "rare event" philosophy of selecting one negative and two positive antigens, we initially tried a "cocktail" of CD45(-)CD56(very bright+) neuron-specific enolase (NSE)(cytoplasmic+). We later modified the procedure to a more clinically applicable "lysed whole blood" CD45(-)CD56(very bright+) ganglioside GD2(+) cocktail to improve turnaround time (eliminating the cell permeabilization step for cytoplasmic NSE analysis), specificity, and sensitivity of the assay. A total of 123 marrow/tissue/fluid samples were analyzed by the various forms of the assay. Clearly interpretable samples had an 83% specificity and a 100% sensitivity. The three-color GD2 assay has successfully detected cells in marrow samples to a level of 0.002% (1 per 10(5) cells) using patient samples (not artificially "spiked" material). We added CD81 expression of the neuroblastoma cells as a fourth color and now use this rare event clinical test to help stage and monitor all patients with neuroblastoma.

Changes in the expression of surface receptors on lymphocyte subsets in the elderly: quantitative flow cytometric analysis.

The immunophenotype of circulating lymphocytes, including the intensity expression of surface receptors, changes with ageing. Until now, no results of systematic studies on age-dependent changes with respect to the expression of the major lymphocyte surface receptors in healthy elderly subjects have been reported. In order to identify age-related changes in both representation and immunophenotype of lymphocyte populations, we investigated, by means of triple-color whole-blood immunostaining and quantitative flow cytometry, the percent values and the absolute numbers, as well as the levels of surface antigen expression or antigen molecules per cell (ABC values x 10(3)), of different peripheral blood lymphocyte subsets from 23 healthy elderly subjects and 13 young donors. Naive (CD45RA+CD3+) T cells, total B cells, and CD5+ B lymphocytes are decreased (22%, 6%, 0.8% vs. 30%, 12%, 1.4%, respectively), whereas activated (HLA-DR+CD3+) and memory (CD45RO+CD3+) T cells, CD3+CD7- T lymphocytes, and lymphocytes expressing the NK marker CD56 are expanded in the elderly (2%, 53%, 13%, 6% vs. 0.8%, 45%, 8%, 8%, respectively). Moreover, T lymphocytes from elderly individuals express lower CD3 (61 +/- 10) compared to young (69 +/- 10). Considering the different T-cell populations, CD3 antigen is respectively decreased on CD45RO+ T cells (55 +/- 14 vs. 66 +/- 14) and up-regulated on CD56+ T lymphocytes (62 +/- 21 vs. 45 +/- 20). Increased CD8 expression characterizes CD3+CD7- lymphocytes (70 +/- 34 vs. 44 +/- 17) while HLA-DR on activated T cells is lower in old (39 +/- 7) than young (46 +/- 9) donors. CD7 is down-regulated both in T (22 +/- 3 vs. 28 +/- 3) and NK (48 +/- 18 vs. 71 +/- 18) cells, whereas CD2 expression, unchanged on NK cells, is up-regulated on T lymphocytes (54 +/- 10 vs. 41 +/- 8). Age-related changes in B-cell antigen expressions were also found: CD20 is increased (124 +/- 23 vs. 105 +/- 16) whereas, despite the unchanged CD5 expression of T cells, CD5 intensity on the B-cell subset co-expressing this antigen is higher in old (49 +/- 37) than in young (22 +/- 4) people. The observed changes in the expression of functionally important cellular receptors can contribute to the remodeling of immune function characteristic of the elderly. Moreover, since quantitative flow cytometry is becoming widely employed in clinical practice, our results also contribute to the assessment of specific age-dependent antigen expression changes to be considered for diagnostic approaches in the elderly.

Quantitative multiparametric immunophenotyping in acute lymphoblastic leukemia: correlation with specific genotype. I. ETV6/AML1 ALLs identification.

The t(12;21)(p13;q22) fusion gene is the most frequent genetic lesion described in precursor B cell acute lymphoblastic leukemia (ALL) of childhood occurring in a quarter of cases. This gene rearrangement is associated with a good outcome presenting a high response rate to chemotherapy. In spite of its potential clinical relevance, the t(12;21) translocation usually goes undetected with conventional cytogenetic procedures. In the present study we utilized an objective flow cytometric approach (multiparametric quantitative analysis) for the phenotypic characterization of this type of ALL. We studied a total of 74 precursor B-ALL children, including 21 t(12;21)+ and 53 t(12;21)- cases. Our results show that the t(12;21)(p13;q22)+ ALLs display a higher intensity of CD10 (P = 0.0016) and HLADR (P = 0.005) expression together with lower levels of the CD20 (P = 0.01), CD45 (P = 0.01), CD135 (P = 0.003) and CD34 (P = 0.03) antigens as compared to the t(12;21) cases. Moreover, as regards CD34 expression, we observed a more heterogeneous antigen expression within individual patients with higher coefficients of variation (median of 202 vs 88, P = 0.0001). A multi-variate analysis disclosed that with the immunophenotypic approach used identification of t(12;21)+ cases can be achieved with a sensitivity of 86% and a specificity of 100%. We conclude that childhood precursor B-ALL carrying the t(12;21) translocation display characteristic phenotypic features which could provide a rapid, simple, sensitive and specific screening method to select for those cases that should undergo confirmatory molecular analysis.

[Immune status studies after one-time alcohol consumption in healthy subjects]. / Untersuchungen zum Immunstatus nach einmaligem Alkoholkonsum bei gesunden Probanden.

AIM OF STUDY: To ascertain whether once-only intake of a large amount of alcohol causes measurable effects on the immune system of healthy persons. SUBJECTS AND METHODS: Cytokine levels, lymphocyte subpopulations and mitogen stimulation were measured in eleven healthy nonalcohol drinking volunteers (eight men, three women; mean age 30.5 [26-36] years; mean weight 71.4 kg) after a single intake of 80 g ethanol (corresponding to 1.12 g/kg). RESULTS: There were no changes in the concentrations of tumour necrosis factor alpha, interleukins 1 alpha and 1 beta, 3, 6, 10, soluble interleukin-6 receptor, interleukin-1 receptor antagonist, gamma-interferon and granulocyte-macrophage colony stimulating factor. A tendency towards an increase was measured regarding the CD-4 lymphocyte counts as well as the concentration of soluble interleukin-2 receptors and intercellular adhesion molecules (ICAM-1). Interleukins 4 and 8 were low normal. Lymphocyte activity was slightly reduced in the mixed lymphocyte cytotoxicity test. No clear-cut changes were observed in the results of the other mitogen stimulation tests. CONCLUSION: These data show that once-only intake of a large amount of alcohol does not produce measurable abnormalities in the immune system of healthy persons who ordinarily abstain from drinking alcohol.

Immunophenotyping of lymphocytes in bronchoalveolar lavage fluid. A new flow cytometric method vs standard immunoperoxidase technique.

Characterizing lymphocyte subsets in bronchoalveolar lavage fluid (BALF) by flow cytometry (FC) proper gating of the lymphocyte subpopulation being analyzed is crucial. In order to test lymphocyte gate quality for the first time we used a DNA-dye to evaluate plasmamembrane integrity and thus to mark off fluorescent but not DNA-containing particles (eg, debris). A comparative prospective study between this newly developed FC technique and a standard peroxidase anti-peroxidase (PAP) method was performed. Samples of BALF from 50 patients with various pulmonary diseases were examined. After determination of the total cell yield, a differential cell count was performed. Subsequently, the immunophenotype of pan T lymphocyte CD3-, T-helper lymphocyte CD4-, and T-suppressor lymphocyte CD8-positive lymphocyte subsets was assessed with FC as well as with the PAP method. Both methods showed excellent correlation (CD3: r = 0.81; CD4: r = 0.97; CD8: r = 0.96; p < 0.05, respectively). Comparing the mean +/- SEM, FC tends to overestimate CD3+ cells (90.6 +/- 1.0% vs 85.8 +/- 1.3%). For CD4 (45.0 +/- 3.4% vs 44.4 +/- 3.4%) and CD8 (48.1 +/- 3.5% vs 46.7 +/- 3.5%), there was good agreement. In a clinical setting, the reliability of both methods was equivalent, and FC using a DNA-dye to test lymphocyte gate quality offered a rapid and reliable determination of lymphocyte subsets in BAL.