Aberrant Antigen Research Articles

Undifferentiated Tumor: True Identity by Immunohistochemistry

"Undifferentiated tumor" refers to a heterogeneous group of neoplasms with little or no evidence of differentiation on routine light microscopic morphology. To identify the true identity of undifferentiated tumors by immunohistochemical analysis. Review of the pertinent literature and the authors' experience. For treatment and prognostic evaluation, it is crucial to delineate whether an undifferentiated neoplasm is epithelial, mesenchymal, melanocytic, or hematopoietic in nature. Application of a screening panel to demonstrate the expression of markers of major lineages is fundamental for determination of the broad category of neoplasia. Because poorly differentiated carcinomas and in particular sarcomatoid carcinomas are known to be heterogeneous in their antigen expression, several epithelial markers in combination may be required to establish the carcinomatous nature of tumor. A diagnostic misinterpretation as a consequence of occasional aberrant or unexpected antigen expression is best avoided by using a broad panel that includes both antibodies that are anticipated to be positive and those that are expected to be negative. In this treatise, the immunohistochemical dissection of undifferentiated tumors on the basis of their morphologic features is outlined, supplemented with algorithmic immunohistochemical analysis for each morphologic category of small round cell tumors, carcinomatous tumors, sarcomatous (or sarcoma-like) tumors, and tumors with histologically overlapping features, including hematolymphoid malignancies, melanoma, and sarcomas with epithelioid appearance. The utility of several organ- or tissue-specific markers in the context of undifferentiated tumors is reviewed.

Aberrant co‐expression of CD19 and CD56 as surrogate markers of acute myeloid leukemias with t(8;21) in Taiwan

Aberrant antigen expression in acute myeloid leukemia (AML) has been extensively studied in the West with limited reports from Taiwan. We carried out this retrospective study to characterize the frequency and significance of aberrant antigen expression of AML in Taiwan. Among 111 cases, 58 (52%) showed aberrant antigen expression, most frequently CD7 (27%) and CD56 (23%). Aberrant CD7 expression was observed in all non-AML-M3 subtypes, most frequently in AML-M7 (4/6, 67%); while CD19 expression was only observed in AML-M2 (5/36, 14%). CD56 expression was most common in AML-M5 (4/8, 50%). The relative frequency of CD19 and CD56 expression in AML with t(8;21) was higher than those with other chromosomal abnormalities or normal karyotype (P = 0.011 and 0.005, respectively). In non-M3 AML, aberrant antigen expression was identified in 56/96 (58%) cases, in contrast to 2/15 (13%) AML-M3 cases (P = 0.001). CD7, CD19 and CD56 expression was not correlated with remission rate. We concluded that aberrant immunophenotype was more frequent in non-M3 leukemias in Taiwan. The relative frequency of CD19 and/or CD56 expression in AML with t(8;21) was significantly higher than those without this translocation and co-expression of these two antigens may serve as the surrogate markers for AML with t(8;21).

Distribution of Antigenic Aberration in the Bone Marrow of Acute Leukemia in Complete Remission

The aberrant, leukemia-associated antigen expression patterns allow us to discriminate leukemic blasts from normal precursor cells. Our major goal was to determine a guideline for the detection of minimal residual disease using CD20+/CD34+ and myeloid Ag+/CD19+ combination in the bone marrow of acute leukemia in complete remission (CR) after chemotherapy. Bone marrow samples from 117 patients with acute leukemia in complete remission after chemotherapy and from 22 healthy controls were immunophenotyped by triple staining and measured by flow cytometry. The CD20+/CD34+ cells in the large lymphocyte gate (R1) ranged from 0% to 3.% (0.8plusmn;0.82%, P=0.000) in CD20+/CD34+ B-lineage ALL CR (N=31), from 0.03% to 4.2% (0.7plusmn;0.83%, P=0.000) in CD20-/CD34- B-lineage ALL CR (N=66), from 0.1% to 0.96% (0.45plusmn;0.32%, P=0.016) in T-ALL CR (N=10), and from 0.02% to 0.48% (0.18plusmn;0.15%, P=0.776) in AML CR (N=10). The CD13,33+/CD19+ cells in R1 gate ranged from 0% to 2.69% (0.37plusmn;0.48%, P<0.001) in CD13,33+/CD19+ B-lineage ALL CR (N=31), from 0% to 1.8% (0.31plusmn;0.28%, P<0.001) in CD13,33-/CD19+B-lineage ALL CR (N=65), from 0.02% to 0.64% (0.29plusmn;0.22%, P=0.071) in T-ALL CR (N=9), and from 0% to 0.17% (0.07plusmn;0.09%, P=0.341) in AML CR (N=3). Using an immunophenotypic method for the detection of early relapse or minimal residual disease of B-lineage ALL bone marrow in CR after chemotherapy, different cutoff values should be applied according to antigen combination and gating. When the proportion of aberrant antigen combination was less than 5% in large lymphocyte gate, the results should be interpreted with caution.

Minimal Residual Disease (MRD) after Frontline Therapy in Adolescent and Young Adult (AYA) Patients with Acute Lymphoblastic Leukemia (ALL) Treated on Two Separate Regimens.

Retrospective and prospective studies have demonstrated that AYA patients (pts) with ALL have superior outcomes when treated with pediatric rather than adult-type regimens. Pediatric ALL regimens are therefore being applied as frontline therapy for adults up to age 30. Given the relatively favorable outcome for this group and the length of therapy employed, it will be several years before the superiority of this approach can be confirmed. MRD by multiparameter flow cytometry (MFC) might be an early measurement of treatment efficacy, since detection of MRD after initial induction has been shown to predict for decreased disease-free survival (DFS). MRD monitoring may thus provide an early indication of later success for different ALL regimens in AYA patients. MRD was measured by four color MFC using lineage specific antibodies in conjunction with antibodies directed at aberrant antigens. The limit of detection was one cell in ten-thousand. MRD data for 43 pts </= 30 years of age treated with the adult regimen of hyper CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone) with or without rituximab was compared with MRD data obtained from 13 pts treated with the pediatric ABFM regimen. The median age of the two groups was 22 years (range 15–30) and 20 years (range 14–28), respectively. MRD data on day 21 of therapy was available for 33 of 43 pts (77%) treated with hyer-CVAD; 22/33 (67%) were negative, 10/33 (30%) were positive, and one pt had insufficient cells for analysis. On day 90 of therapy, 21 of the 43 pts had data available, and all were MRD negative. All pts treated with the ABFM protocol had MRD assessments performed on day 29 of induction therapy; 8 (62%) were negative, 2 (15%) had suspicious cells in an insufficient sample, and 3 (23%) were positive. Of the 9 pts with MRD measurements on day 84 of therapy, all were negative except for 1 pt who relapsed before day 84. These preliminary data indicate that AYA pts with ALL receiving either hyper-CVAD or ABFM therapy have similar MRD status at the end of induction and after approximately 3 months of consolidation. Given the likely importance of MRD in predicting DFS, this comparison suggests that hyper-CVAD and ABFM may show similar outcomes in this population. Continued accrual and longer follow-up will be required to confirm this hypothesis.

Detection of Dysplastic Features by Multiparameter Flow Cytometry in Myelodysplastic Syndromes in Relation to Cytomorphology and Cytogenetics.

Dysplastic features can be detected in different cell lineages in myelodysplastic syndromes (MDS) by multiparameter flow cytometry (MFC). The aim of the present study has been the assessment of the flow cytometric detection of dysplastic features previously published to occur in MDS in relation to findings in cytomorphology (CM) and cytogenetics (CG). We analyzed 307 bone marrow samples from patients with suspected (n=130) or proven (n=177) MDS by MFC, CM, and CG in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 3.5%) and from 0% to 23% (median, 3%; r=0.271, p<0.0001). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 5), 1 for monocytes (0 to 5), and 0 for erythroid cells (0 to 2); median total number=3, range 0–11. The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (13.7%), CD15 (10.4%) and CD64 (10.4%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (67.1%), aberrant coexpression of CD56 (32.9%), aberrant pattern of CD13/CD16 expression (31.6%), aberrant pattern of CD11b/CD16 expression (24.1%), and reduced expression of CD33 (13.4%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (42.7%) and of CD16 (18.9%). The most frequent dysplastic features observed in the erythroid cell populations included a lack of CD71 expression (15.0%) and an aberrantly homogeneous expression of CD71 (9.1%). As compared to cases with no indication of MDS by CM (=non-MDS) cases with MDS according to CM were significantly associated with a reduced side-scatter signal in granulocytes (ratio granulocytes:lymphocytes 6.53±1.27 vs. 7.44±1.17, p<0.0001) as well as a higher number of dysplastic features in granulocytes (1.98±1.09 vs. 1.00±1.31, p<0.0001), monocytes (0.81±0.84 vs. 0.35±0.63, p<0.0001), and erythroid cells (0.33±0.47 vs. 0.20±0.40, p=0.061). Particularly, an aberrant expression of CD56 in monocytes occurred more frequently in 33 cases with CMML as compared to non-MDS cases (84.8% vs. 15.7%, p<0.0001). In cases with possible MDS according to CM the differences to non-MDS cases were less pronounced (reduced side-scatter signal in granulocytes 7.77±1.47 vs. 7.44±1.17, n.s., dysplastic features in granulocytes 1.90±1.21 vs. 1.00±1.31, p=0.003, monocytes 0.50±0.63 vs. 0.35±0.63, n.s., and erythroid cells 0.30±0.47 vs. 0.20±0.40, n.s.). In cases with aberrant cytogenetics (n=80, excluding those with –Y as sole aberration) dysplastic features by MFC occurred more frequently as compared to cases with normal karyotypes (reduced side-scatter signal in granulocytes 6.35±1.18 vs. 7.09±1.38, p<0.0001, dysplastic features in granulocytes 2.05±0.95 vs. 1.68±1.31, p=0.021, monocytes 0.80±0.89 vs. 0.71±0.81, n.s., and erythroid cells 0.38±0.49 vs. 0.31±0.48, n.s.). A total of more than two dysplastic features in blasts, granulocytes, and monocytes and/or a blast count >5% occurred in 68.4%, 63.3%, and 35.3% of cases with MDS, possible MDS, and non-MDS according to CM (p=0.0005). Interestingly, in some cases aberrant antigen expression has been observed in cell lineages not rated dysplastic by CM. This evaluation suggests that MFC may be used to identify dysplastic features in patients with suspected MDS. Sensitivity and specificity varies between MDS subtypes and should be clearly defined in future studies.

The Combination of Fludarabine and Cyclophosphamide Has a Beneficial Effect on the Incidence of Hemolytic Anemia in Chronic Lymphocytic Leukemia. Results from the UK LRF CLL4 Trial.

Autoimmune hemolytic anemia (AHA) is a common complication in chronic lymphocytic leukemia (CLL), occurring in 10–25% of patients during the course of their disease. In 1966 it was first suggested that treatment with X-rays or alkylating agents might trigger the onset of AHA in CLL. Following the advent of purine analog therapy in the 1980s there have been case reports and small series, involving over 100 patients, suggesting that AHA may occur more commonly following treatment with these agents. The UK LRF CLL4 trial is the largest prospective trial in CLL to examine the risk of developing both a positive direct antiglobulin test (DAT) and AHA during treatment. 777 patients were randomized to receive chlorambucil or fludarabine alone or with cyclophosphamide (FC). The incidence of a positive DAT was 14% and AHA 10%. The DAT correctly predicted the development, or not, of AHA after therapy in 83% of cases. The approximate risk of a patient with a positive DAT developing AHA was 1 in 3. Of 299 patients tested both pre- and post-treatment, those treated with single-agent fludarabine were most likely to remain DAT positive and to change from negative to positive. Patients treated with chlorambucil or fludarabine were more than twice as likely to develop AHA as those receiving FC (12%, 11% and 5% respectively). In a multivariate analysis, age, DAT result, stage C disease and treatment were independent predictors of AHA. Patients who developed AHA were less likely than others to respond to treatment (66% versus 81% overall response rate, p=0.004) and had both shorter progression free survival (PFS) (9% versus 18% at 5 years: Odds ratio (OR)=0.55 95%CI:0.40–0.75, p=0.0005) and shorter overall survival (OS) (37% versus 58% at 5 years:OR= 0.4, 95%CI 0.25–0.3, p=0.0004). A positive DAT, with or without AHA, was also associated with shorter PFS and OS. The mechanism of AHA in CLL is still poorly understood. It usually results from production of high affinity polyclonal IgG auto-antibodies by the non-malignant B- lymphocytes. Possible mechanisms include: aberrant antigen presentation by CLL B-cells, defective T-cell function, and loss of regulatory T-cells, with subsequent failure of control of auto-reactive T-cells. The latter may result from initiation of therapy and explain the frequent reports of AHA occurring in association with treatment for CLL. In LRF CLL4 the lowest incidence of AHA (5%) was seen in the FC arm. Possible explanations for this include: the better responses seen in the FC arm which resulted in more rapid and effective control of CLL, the lower total dose of fludarabine administered in FC compared to fludarabine mono-therapy, and the possible protective effect of cyclophosphamide, an immunosuppressive agent, when administered in combination with fludarabine. In conclusion, the FC combination has a beneficial effect, with less likelihood that patients will develop AHA during treatment. Patients with a positive DAT should receive this regimen in preference to mono-therapy. In addition, both DAT status at the time of initiation of therapy and the development of AHA during therapy provide new prognostic indicators for both PFS and OS in CLL.

Aberrant phenotypes and quantitative antigen expression in different subtypes of canine lymphoma by flow cytometry

Flow cytometry may be a useful tool to analyze lymphoma samples that are obtained from fine needle aspirations (FNA). This study aimed to determine if flow cytometric analysis add more objective and standardized information on the cellularity and morphology of lymphoma cells to conventional cytology. The typical immunophenotype of different lymphoma subtypes was assessed and leukocyte marker expression was evaluated to determine which antigens were more frequently over- or under-expressed in these lymphoma subtypes. Fifty FNA lymph node samples were evaluated from canine lymphomas. Thirty-one samples were identified to be of B-cell origin, sixteen were identified to be of T/NK-cell origin and three cases were classified as acute lymphoblastic leukaemia with lymph nodes involvement. The most common B-cell lymphoma subtypes were centroblastic lymphomas, whereas three cases were atypical and classified as B-large cell pleomorphic lymphomas. Among the T/NK lymphomas, small clear cells, large and small pleomorphic mixed cells, large granular lymphocytic cells and small pleomorphic cells were identified. Aberrant phenotypes and/or antigen under/over regulation was identified in thirty out of forty-seven lymphoma cases (64%; 18/31 B-cell = 58% and 12/16 T-cell = 75%). In B-cell lymphomas the most frequent finding was the diminished expression of CD79a (45%). CD34 expression was also observed in four cases (13%). Among T-cell lymphomas the prevalent unusual phenotype was the under-expression or absence of CD45 (25%). These findings reveal flow cytometry may be useful in confirming the diagnosis of lymphoma, as the technique allows one to add useful information about morphology of the neoplastic cells and identify antigenic markers and aberrant phenotypes.

De novo autoimmune hepatitis after liver transplantation

The Kings College group was the first to describe a clinical syndrome similar to autoimmune hepatitis in children and young adults transplanted for non-immune mediated liver diseases. They coined the term "de novo autoimmune hepatitis". Several other liver transplant centres confirmed this observation. Even though the condition is uncommon, patients with de novo AIH are now seen in most of the major transplant centres. The disease is usually characterized by features of acute hepatitis in otherwise stable transplant recipients. The most characteristic laboratory hallmark is a marked hypergammaglobulinaemia. Autoantibodies are common, mostly ANA. We described also a case of LKM1-positivity in a patients transplanted for Wilson's disease, however this patients did not develop clinical or histological features of AIH. Development of SLA/LP-autoantibodies is also not described. Therefore, serologically de novo AIH appears to correspond to type 1 AIH. Like classical AIH patients respond promptly to treatment with increased doses of prednisolone and azathioprine, while the calcineurin inhibitors cyclosporine or tacrolimus areof very limited value - which is not surprising, as almost all patients develop de novo AIH while receiving these drugs. Despite the good response to treatment, most patients remain a clinical challenge as complete stable remissions are uncommon and flares, relapses and chronic disease activity can often occur. Pathogenetically this syndrome is intriguing. It is not clear, if the immune response is directed against allo-antigens, neo-antigens in the liver, or self-antigens, possibly shared by donor and host cells. It is very likely that the inflammatory milieu due to alloreactive cells in the transplanted organ contribute to the disease process. Either leading to aberrant antigen presentation, or providing co-stimulatory signals leading to the breaking of self-tolerance. The development of this disease in the presence of treatment with calcineurin inhibitors supports the view held by most specialists in autoimmune hepatitis that these drugs, even though effective in acute disease, are not helpful in the long-term management of autoimmune liver diseases.

Aberrant immunophenotype in acute myeloid leukemia (AML): High frequency and association with dysplasia

17504 Background: Asynchronous expression of immunophenotypic markers on AML myeloblasts has been well described, but the association of aberrant phenotype with morphologic subclasses has not been reported previously. Methods: Multiparameter flow cytometry (MFC) data were analyzed for all patients (pts) diagnosed with AML at our institution from 2000–2006. MFC was done on fresh bone marrow aspirate and/or peripheral blood samples using the following panel of monoclonal antibodies in triple staining: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD24, CD33, CD56, CD34, CD117, HLA-DR and TdT. Identification of myeloblasts was performed using Side Scatter (SS) characteristics and CD45 expression. Acute lymphoblastic leukemia (ALL) cases or cases with mixed lineage acute leukemia were excluded from the study. Results: We identified 76 pts with AML; median age 57, (range 10 weeks to 89 years), with 32 males and 44 females. The patients had the following French American British classification of AML: M0 (n=1), M1 (n=11), M2 (m=10), M3 (n=5), M4 (n=13), M5 (n=14), M6 (n=1), M7 (n=4) and not otherwise categorized non- M3 AML (n=17). Twenty two pts had AML with multilineage dysplasia (AML-MD) as classified by World Health Organization. Coexpression of TdT on myeloblasts was observed in 4 pts, one of these coexpressed CD5. T-cell lymphoid associated antigens (CD2, CD5 and/or CD7) were coexpressed on myeloblasts from12 pts. B cell lymphoid associated antigens (CD19 and/or CD20) were expressed on myeloblasts from11 pts. Both T and B cell lymphoid associated antigens were present on myeloblasts in 4 pts. Myeloblasts from 13 pts demonstrated the presence of the NK cell marker CD56. A total of 43 patients had AML with immunophenotypic lineage infidelity manifested by coexpression of one or more aberrant antigens on myeloblasts. The distribution of cases with lineage infidelity was higher in 22 pts with AML-MD (n=17, 77%) versus in 54 pts with no dysplasia (n=26, 48%), Chi square =5.4, p=0.02. Conclusions: Abnormal coexpression of lymphoid and NK cell markers was observed in significant number of AML pts and correlated with the presence of multilineage dysplasia. This finding may indicate an earlier stem cell origin of AML-MD. No significant financial relationships to disclose.

Aberrant Leukocyte Common Antigen Expression in Metastatic Small Cell Lung Carcinoma: A Rare Finding and a Potential Diagnostic Pitfall

Aberrant Leukocyte Common Antigen Expression in Metastatic Small Cell Lung Carcinoma: A Rare Finding and a Potential Diagnostic Pitfall

Anaplastic lymphoma kinase–positive diffuse large B-cell lymphoma with a complex karyotype and cryptic 3′ ALK gene insertion to chromosome 4 q22-24

Anaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (DLBCL) is a rare tumor that is frequently associated with t(2;17)(p23;q23), a translocation fusing the ALK gene at 2p23 to the clathrin heavy chain gene (CLTC) at 17q23. Here, we report a unique case of ALK-positive DLBCL with plasmablastic morphology and focal cytoplasmic granular ALK stain in an HIV-negative 33-year-old man. By conventional karyotyping, the lymphoma cells were near-tetraploid and included 4 structurally normal copies each of chromosomes 2 and 17. Fluorescence in situ hybridization revealed an apparently normal, intact ALK gene on each of the 4 chromosome 2 homologs plus a cytogenetically cryptic ALK gene insertion into 2 of the 4 chromosome 4 homologs at band 4q22-24. The lymphoma cells expressed CD138, lambda light chain, focal and weak CD30, and exhibited aberrant T-cell antigens, including perforin. This case indicates that ALK-positive DLBCL is more heterogeneous at the cytogenetic/molecular level than previously recognized.

A comprehensive approach to the diagnosis of MDS after triage by morphology towards cytogenetics and other techniques

The myelodysplastic syndromes (MDS) are heterogeneous disorders ranging from rather stable conditions such as 5q- to advanced stages close to acute leukaemia. The variability of the clinical course, of cytomorphological phenotypes, and of genetic aberrations outlines MDS classification as a major challenge. However, therapeutic options became more differentiated e.g. due to lenalidomide or reduced intensity conditioning in allogeneic transplantation and require a more detailed risk stratification. Established classification systems as the FAB were mainly based on cytomorphological criteria implicating some problems e.g. of interobserver variability. The WHO proposal of 2001 which subdivided MDS into 10 subtypes focused as well mainly on cytomorphology, but took in addition cytogenetics with respect to the 5q- syndrome into account. Another important classification system is represented by the International Prognostic Scoring System (IPSS) of 1997 which offers an accurate biologically based classification and prognostication. Multiparameter flow cytometry (MFC) and molecular techniques may contribute increasingly by detection of aberrant antigen expression patterns and by the finding that some mutations e.g. the FLT3-LM/ITD or NRAS are associated with leukaemic transformation. In conclusion, a multimodal approach including cytomorphology, cytogenetics, fluorescence in situ hybridization (FISH), PCR, and MFC is possible to achieve a comprehensive characterization of MDS. Diagnostic algorithms providing a hierarchy of methods to guarantee an efficient work-flow, to provide all parameters necessary for classification and for minimal-residual-disease (MRD) strategies, and to guide new treatment strategies are needed.

Aberrant Megakaryocyte Antigen Expression on Myelodysplastic Erythroid Cells: Role of Protein Kinase C?.

Erythroid lineage dysplasia is one of the most frequent findings in Myelodysplastic Syndrome (MDS) and in clinical terms contributes significantly to the morbidity in affected patients. There is currently no satisfactory treatment available for the majority of these patients and many rely on regular red cell transfusions for support. Previous work using an in vitro model system based on human CD34+ progenitors indicated that the developmental abnormalities affecting the erythroid cells may in part arise from inappropriate activation of protein kinase C (PKC). Evidence suggests that PKC acts as a lineage discriminator for bipotential erythro-megakaryocytic progenitor cells where low PKC activity promotes erythroid commitment and high PKC activity promotes megakaryocytic commitment. In the in vitro model system aberrant activation of PKC in erythroid cells led to developmental disruption and inappropriate megakaryocytic antigen expression such as GPIIb (CD41). Restoration of normal development was possible by inhibition of PKC. In this study we investigate the extent to which aberrant megakaryocyte antigen expression occurs in MDS, and whether PKC inhibition can normalise this and the associated erythroid lineage dysplasia. Bone marrow was obtained from 27 MDS patients and compared with 16 normal bone marrows. Fresh bone marrow mononuclear cells from both groups were analysed by 4- colour flow cytometry to establish the frequency of aberrant CD41 expression on nucleated erythroid (GlyA+ CD36+) cells. Cells bearing adherent platelets were excluded based on co-expression of CD42b. We have established that aberrant expression of CD41 occurred in 56% of the MDS patient marrows compared with normal bone marrow erythroid cells (n=27 and 16 respectively). These patients also showed clear morphological evidence of erythro-megakaryocytic lineage dysplasia. To determine the effect of PKC inhibition on this aberrant CD41 expression, some of these cases were examined in bulk liquid culture in the presence of PKC inhibitors. We assessed three PKC inhibitors in this way: GF109203X, a proprietary staurosporine derivative and Tamoxifen. In 42% of the cases examined in this way there was a phenotypic response to the highly selective PKC inhibitor GF109203X in terms of a reduction in CD41 expression. There was also morphological evidence of erythroid differentiation. The other two agents showed no effect on these parameters. The expression profile of individual PKC isoforms in the erythroid (GlyA+) cells was examined by western blotting, to determine if as in the model system the developmental abnormalites seen in the MDS marrow resulted from hyperactivation of PKC. We were able to evaluate 13 MDS and 6 normal controls in this way. The expression of 3 PKC isoforms (α, ε, ι) was determined as well as the level of phosphorylated PKC and the level of PDK1 kinase (which phosphorylates PKC). More than half of the patients examined demonstrated elevated levels of PKC expression. Aberrations in phosphorylation of PKC were less common. Patients with the highest expression of CD41 also had the highest expression of PKCα. These data suggest a role of inappropriate PKC activity in the erythroid lineage dysplasia and subsequent developmental failure seen in MDS, and, that PKC inhibitors may be valuable therapeutic agents in a subset of these patients.

Aberrant Receptor-Mediated Endocytosis of Schistosoma mansoni Glycoproteins on Host Lipoproteins

BackgroundBilharzia is one of the major parasitic infections affecting the public health and socioeconomic circumstances in (sub) tropical areas. Its causative agents are schistosomes. Since these worms remain in their host for decades, they have developed mechanisms to evade or resist the immune system. Like several other parasites, their surface membranes are coated with a protective layer of glycoproteins that are anchored by a lipid modification.Methods and FindingsWe studied the release of glycosyl-phosphatidylinositol (GPI)-anchored proteins of S. mansoni and found them in the circulation associated with host lipoprotein particles. Host cells endocytosed schistosomal GPI-anchored proteins via their lipoprotein receptor pathway, resulting in disturbed lysosome morphology. In patients suffering from chronic schistosomiasis, antibodies attacked the parasite GPI-anchored glycoproteins that were associated with the patients' own lipoprotein particles. These immunocomplexes were endocytosed by cells carrying an immunoglobulin-Fc receptor, leading to clearance of lipoproteins by the immune system. As a consequence, neutral lipids accumulated in neutrophils of infected hamsters and in human neutrophils incubated with patient serum, and this accumulation was associated with apoptosis and reduced neutrophil viability. Also, Trypanosoma brucei, the parasite that causes sleeping sickness, released its major GPI-anchored glycoprotein VSG221 on lipoprotein particles, demonstrating that this process is generalizable to other pathogens/parasites.ConclusionsTransfer of parasite antigens to host cells via host lipoproteins disrupts lipid homeostasis in immune cells, promotes neutrophil apoptosis, may result in aberrant antigen presentation in host cells, and thus cause an inefficient immune response against the pathogen.

A Pilot Study Evaluating the Safety and Efficacy of Imatinib Given Early after Transplant for High-Risk Philadelphia Chromosome-Postive Leukemias.

Patients with Ph+ acute lymphoblastic leukemia (ALL) or chronic myelogenous leukemia (CML) in stages other than first chronic phase (CP1) frequently have recurrent malignancy after allogeneic hematopoietic cell transplant (HCT). Imatinib given after HCT for the treatment of hematological relapse has been of limited success in Ph+ALL but may induce more durable remissions in CML.Hypothesis: We postulated that imatinib might be most effective for preventing hematological relapse after myeloablative HCT if given immediately after engraftment to patients without detectable leukemia, or with leukemia that can be detected only at the molecular level.Study design: A pilot study is ongoing to evaluate the safety and preliminary efficacy of imatinib begun early after myeloablative HCT and continued until post-transplant day 365 (D+365). Study participants became eligible to start imatinib (adults 400 mg/day, children 260 mg/m2/day) if the residual marrow leukemia burden at the time of initial engraftment (ANC>500 on 2 consecutive days) did not exceed >1/20 Ph+ metaphases, >1% aberrant antigen expression on blasts by multidimensional flow, or presence of bcr/abl in >5% interphase nuclei by FISH. The primary endpoint of safety was defined by ability to tolerate imatinib (adults ≥200 mg/day, children ≥100 mg/day) for ≥ 6 days/week until D+90. An attempt was made to administer higher daily doses of imatinib after D+90.Patient characteristics: Ten patients with Ph+ALL (8 CR1, 2 CR2) and 6 patients with CML (2 AP, 2 CP2, 2 CP3) have been enrolled; 13/16 had leukemia detected by molecular or cytogenetic methods at the time of transplant. Median age at transplant was 40 y (range 5–62 y). Stem cell sources were cord blood (n=1), marrow (n=4) or G-mobilized peripheral blood (n=11). Donors were unrelated (n=10) or related (n=6).Results: Imatinib therapy began in 15 patients at a median of 29 days (range 24–39 days) after HCT and has been administered for a median of 299 days (range, 33–380 days). The median of average daily doses during this time period was 400 mg/day (range 389 to 510 mg/day) among adults and 304 mg/m2/day for the 2 children. All patients tolerated imatinib at the intended dose intensity within the first 90 days after HCT. Toxicities (NCI CTC v3.0) possibly attributed to imatinib included grade 1–2 nausea (n=3), grade 1 edema (n=3), grade 1–2 anemia (n=2), and grade 3 neutropenia (n=2). Per protocol, one patient with neutropenia received 2 doses of G-CSF at D+75 and continued imatinib without neutropenia. The second patient was not given G-CSF and imatinib was held for 2 weeks from D+160 until the ANC was >2000. All patients are surviving at a median of 333 days after HCT (range, 68–564), and 14/15 patients have no detectable bcr/abl transcripts in the blood or marrow. Seven patients (4 ALL, 3 CML) have completed imatinib therapy and survive at a median of 467 days after HCT (range, 410–564 days) and 6/7 have no detectable bcr/abl transcripts in blood or marrow. One patient (CML-CP3) with cytogenetic relapse at D+118 had a 4th remission after withdrawal of immunosuppression and continued imatinib but developed hematological relapse at D+429.Conclusions: We conclude that imatinib therapy can be safely prescribed early after myeloablative allogeneic HCT at a dose-intensity comparable to that used in general oncology. Preliminary efficacy data are encouraging and worthy of further study.

Flow Cytometric Characteristics of CD34+ Cells in Refractory Anemia: Their Diagnostic Value.

(INTRODUCTION) The diagnosis of refractory anemia (RA), a subtype of myelodysplastic syndromes (MDS), is often problematic because RA often does not show cytogenetic abnormalities and dysplastic hematopoietic cells are not specific to MDS. Recent studies have suggested that the abnormalities of hematopoietic cells detected by flow cytometry (FCM) might be useful for MDS diagnosis. However, this concept has not yet been widely accepted. We reported that MDS blasts often show aberrant antigen expression. In this study, we examined the diagnostic utility of the flow cytometric characteristics of CD34+ cells in RA.(METHODS) Whole bone marrow cells obtained from RA patients and controls (e.g., cytopenic individuals other than RA and other malignancies) were analyzed using four-color FCM. The cells were stained with CD45 and CD34, for gating CD34+ B-cell precursors (hematogones) and CD34+ myeloblasts, in combination with various other antibodies. We determined percentages of hematogones and CD34+ myeloblasts and relative mean florescence intensity (RMFI: MFI of each antigen staining ÷ MFI of isotype-matched negative control staining) of various antigens on CD34+ myeloblasts.(RESULTS) From the data on 50 control subjects in the first subject cohort, we determined the reference range (RR) for each flow cytometric parameter. We scored data on each subject by giving 1 point to each of 10 parameters if the data of such parameters were outside RRs. These 10 parameters, which were adopted after comparing data from control and RA subjects in the first cohort, were percentages of hematogones and CD34+ myeloblasts and RMFI of CD4, CD56, CD11b, CD13, CD15, CD33, CD117 and CD133. In the first cohort, scores of all control subjects were less than 3 (40 subjects had a score of 0) and scores of 7/12 RA patients were 3 or higher. Using the same parameters and same RRs, we then prospectively analyzed the second subject cohort. Again, this approach identified 8/13 RA patients (score 3 or higher) with 100% specificity. The degree of dysplasia and presence or absence of cytogenetic abberations were not related to RA identification.(CONCLUSION) Our method is reproducible and helpful in RA diagnosis.

Flow Cytometric Analysis of Monocytes as a Tool for Distinguishing Chronic Myelomonocytic Leukemia From Reactive Monocytosis

To determine whether immunophenotypic features of monocytes are useful in differentiating chronic myelomonocytic leukemia (CMML) from reactive monocytosis, multiparameter flow cytometry was used to immunophenotype 20 bone marrow samples from patients with CMML, 10 normal marrow samples, and 20 marrow samples with reactive monocytosis. Monocytes in CMML exhibited aberrant antigen expression in all 20 cases. Abnormal antigen expression also was observed in monocytes in 11 of 20 reactive marrow samples. However, aberrant expression of 2 or more antigens was significantly less frequent in reactive monocytosis than in CMML (P = .002). CD56 expression with underexpression of a myeloid marker was unique to CMML monocytes. Subpopulations of monocytes with moderate levels of CD14 were present in all 3 groups. The proportion of CD14(moderate) monocytes was highest in CMML and was 20% or more in 13 of 20 CMML cases vs 3 of 20 reactive marrow samples (P = .003) and 2 of 10 normal marrow samples (P = .007). A combination of monocytosis with 2 or more immunophenotypic aberrancies with 20% or more of marrow monocytes showing moderate CD14 expression was 67% sensitive and 100% specific for CMML.

Flow Cytometric Analysis of Monocytes as a Tool for Distinguishing Chronic Myelomonocytic Leukemia From Reactive Monocytosis

To determine whether immunophenotypic features of monocytes are useful in differentiating chronic myelomonocytic leukemia (CMML) from reactive monocytosis, multiparameter flow cytometry was used to immunophenotype 20 bone marrow samples from patients with CMML, 10 normal marrow samples, and 20 marrow samples with reactive monocytosis. Monocytes in CMML exhibited aberrant antigen expression in all 20 cases. Abnormal antigen expression also was observed in monocytes in 11 of 20 reactive marrow samples. However, aberrant expression of 2 or more antigens was significantly less frequent in reactive monocytosis than in CMML (P = .002). CD56 expression with underexpression of a myeloid marker was unique to CMML monocytes. Subpopulations of monocytes with moderate levels of CD14 were present in all 3 groups. The proportion of CD14(moderate) monocytes was highest in CMML and was 20% or more in 13 of 20 CMML cases vs 3 of 20 reactive marrow samples (P = .003) and 2 of 10 normal marrow samples (P = .007). A combination of monocytosis with 2 or more immunophenotypic aberrancies with 20% or more of marrow monocytes showing moderate CD14 expression was 67% sensitive and 100% specific for CMML.

Dendritic cell dysfunction in cancer: A mechanism for immunosuppression

Several reports have demonstrated that tumours are not intrinsically resistant to the immune response. However, neoplasias commonly fail to initiate and maintain adequate immunity. A number of factors have been implicated in causing the failure, including aberrant antigen processing by tumour cells, anergy or deletion of T cells, and recruitment of inhibitory/regulatory cell types. It has been suggested that dysfunction of dendritic cells (DC) induced by the tumour is one of the critical mechanisms to escape immune surveillance. As a minor subset of leucocytes, DC are the key APC for initiating immune responses. DC are poised at the boundaries of the periphery and the inner tissues, sampling antigens of diverse origin. Following their encounter with antigen or danger signals, DC migrate to lymph nodes, where they activate effector cells essential for tumour clearance. Although the DC system is highly heterogeneous, the differentiation and function of DC populations is largely regulated by exogenous factors. Malignancies appear to exploit this by producing a plethora of immunosuppressive factors capable of affecting DC, thus exerting systemic effects on immune function. This review examines recent findings on the effects of tumour-derived factors inducing DC dysfunction and in particular examines the findings on alteration of DC differentiation, maturation and longevity as a potent mechanism for immune suppression in cancer.

Application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression

To investigate the application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression. The results of flow cytometry of 30 lymphoma/leukemia cases with aberrant antigen expression, of which 3 cases being lymphomas, 8 B-cell leukemia, 1 T-cell leukemia, 17 acute non-lymphoid leukemia and 1 acute non-lymphoid leukemia involving lymph nodes were analyzed. Immunohistochemistry (EnVision) for CD79a, CD3 and MPO was performed on all cases. Eleven cases of B-cell lymphoma/leukemia were cytoplasmic CD79a (cCD79a)-positive, cytoplasmic CD3 (cCD3epsilon) and cytoplasmic MPO (cMPO)-negative. Five of these cases were positive for CD5 and 2 for CD5, 1 or 2 for myeloid marker(s). The T-cell leukemia cases were cCD3epsilon-positive, cCD79a and cMPO-negative, they also co-expressed CD13 and CD33. The mantle cell lymphoma cases were positive for CD3, CD13 and CD33. Of the 8 B-cell leukemia cases, 4 were positive for CD5, 3 for CD13 and 1 for CD13 and CD33. The 18 acute non-lymphoid leukemia cases (including 1 acute non-lymphoid leukemia case involving lymph nodes) were cMPO-positive and cCD79a and cCD3epsilon-negative. Eight of the 18 expressed T-cell markers (including 1 case of acute non-lymphoid leukemia involving lymph nodes), 8 expressed B-cell markers, 2 expressed both T and B-cell markers. Flow cytometry can demonstrate aberrant antigen expression in lymphoma/leukemia cells and is helpful in delineating their cell origin. The technique is thus useful in the differential diagnosis of lymphoma/leukemia.