Aberrant Expression Of CD56 Research Articles

Urticaria pigmentosa and mastocytosis: The role of immunophenotyping in diagnosis and determining response to treatment

Recent advances in our understanding of mast cell biology and disease resulted in identification of important differences in expression of mast cell surface antigens in normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts leads to the inclusion of this immunophenotypic abnormality in the World Health Organization's diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients with limited disease that lacks histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. Flow cytometric analysis of bone marrow mast cells is therefore a sensitive method of diagnosis of mast cell disease and is expected to find increasing use in determining response to emerging mast cell cytoreductive therapies.

Immunophenotyping of Plasma Cells

Plasma cell enumeration and immunophenotyping has been shown to be of value in predicting the outcome for patients with myeloma. Detection of abnormal plasma cells at a leukocyte level >0.01% at the end of therapy predicts early relapse. Although clonally related populations at varying stages of differentiation are reported to be present in myeloma, only the CD138+ fraction has been shown to directly correlate with the outcome. In addition, the plasma cell immunophenotype in monoclonal gammopathy of undetermined significance (MGUS) is reported to correlate with the risk of progression to myeloma. This protocol provides a four-color flow cytometry method for enumeration and classification of plasma cells in both diagnostic and post-treatment samples. Plasma cells are identified by strong expression of CD38 and CD138. Neoplastic plasma cells are separated from their normal counterparts by aberrant expression of CD19 and other relevant markers in combination with intracellular immunoglobulin light chain restriction.

Atypical NK-cell Proliferation of the Gastrointestinal Tract in a Patient With Antigliadin Antibodies but not Celiac Disease

We describe a unique case of atypical natural killer (NK)-cell proliferation likely related to gluten sensitivity, mimicking NK-cell lymphoma. The patient, a 32-year-old man, has had persistent multiple erythematous bull-eye lesions in the stomach, small bowel, and large bowel for 3 years. Histologically, the lesions were well circumscribed and relatively superficial, composed of atypical medium-sized to large-sized lymphocytes with slightly irregular nuclear contours, a dispersed chromatin pattern, and clear cytoplasm. Immunohistochemistry and flow cytometry showed that the cells were NK cells expressing CD56 (aberrantly bright), T-cell intracellular antigen (TIA)-1, cytoplasmic CD3, and CD94, but not surface CD3, with bright aberrant expression of CD7 and a lack of other NK cell-associated markers. Polymerase chain reaction for rearrangement of the T-cell receptor-gamma chain gene showed no evidence of a clonal T-cell population, and in situ hybridization for Epstein-Barr virus encoded RNA was negative. There was no evidence of the involvement of peripheral blood or bone marrow. Although a diagnosis of extranodal NK/T-cell lymphoma was considered because of the atypical morphology and immunophenotypic aberrancy, no chemotherapy was given because of the relatively superficial nature of the infiltrates, lack of significant symptoms, and negativity for Epstein-Barr virus. Two years after initial presentation, the patient was found to have high titers of antigliadin antibodies with no other evidence of celiac disease. After instituting a gluten-free diet, many of the lesions regressed, suggesting that this atypical NK-cell proliferation may be driven by an anomalous immune response. Awareness of this case may prevent pathologists from misdiagnosing similar lesions as NK/T-cell lymphomas. It is as yet unknown whether this process occurs more commonly in patients with gluten sensitivity, or in other settings, and the pathogenesis is as yet undetermined.

CD10 expression in peripheral T-cell lymphomas complicated by a proliferation of large B-cells

CD10 expression by the neoplastic T cells in angioimmunoblastic T-cell lymphoma was recently described. As cases of peripheral T-cell lymphoma, unspecified, fail to show similar CD10 expression, this feature helps discriminate between these two entities, particularly in cases exhibiting morphologic overlap. Given these findings, we studied CD10 expression in a subtype of peripheral T-cell lymphoma known as peripheral T-cell lymphoma complicated by a proliferation of large B cells and compared it with angioimmunoblastic T-cell lymphoma and angioimmunoblastic T-cell lymphoma with a large B-cell proliferation. A total of 33 cases were identified including peripheral T-cell lymphoma complicated by a proliferation of large B cells (10), angioimmunoblastic T-cell lymphoma (10) and angioimmunoblastic T-cell lymphoma with a large B-cell proliferation (13). Diagnoses were established by hematoxylin and eosin (H&E) stain, immunohistochemistry and/or molecular findings (polymerase chain reaction for T-cell receptor-gamma gene rearrangement). Two of 10 cases of peripheral T-cell lymphoma complicated by a proliferation of large B cells showed aberrant CD10 expression (20%) compared to 9/10 cases of angioimmunoblastic T-cell lymphoma (90%) and 8/13 of angioimmunoblastic T-cell lymphoma with a large B-cell proliferation (62%). One case each of angioimmunoblastic T-cell lymphoma and angioimmunoblastic T-cell lymphoma with a large B-cell proliferation showed a rare, but not unequivocal, CD10(+) atypical cell. Four cases of angioimmunoblastic T-cell lymphoma with a large B-cell proliferation were CD10 negative. Of the 2 CD10(+) peripheral T-cell lymphoma complicated by a proliferation of large B cells, one had no H&E or IHC features of angioimmunoblastic T-cell lymphoma and showed only a rare positive cell. The second case, a lung biopsy, exhibited diffuse CD10 tumor cell positivity. The predominant staining pattern in the CD10(+) cases was characterized by scattered, mostly individual, morphologically neoplastic cells. A rare case showed clusters of positive cells. Our data indicate that only 20% of cases of peripheral T-cell lymphoma complicated by a proliferation of large B cells show CD10 expression by the neoplastic T cells in contrast to angioimmunoblastic T-cell lymphoma and angioimmunoblastic T-cell lymphoma with a large B-cell proliferation which exhibit CD10 staining in 90 and 62% of cases, respectively. This finding does not reach statistical significance with a P-value of 0.57 (Fisher's exact test). As these entities appear to be biologically distinct and may portend different overall survivals, CD10 expression may serve as an additional discriminating criterion.

Angioimmunoblastic T- Cell Lymphoma by Flow Cytometric Analysis.

Background: Angioimmunoblastic T cell lymphoma (AILT) is a peripheral T- cell lymphoma characterized by systemic disease, a polymorphous infiltrate involving lymph nodes with prominent proliferation of high endothelial venules and follicular dendritic cells. We present a novel technique for the presumptive diagnosis of AILT cases by flow cytometric analysis. The diagnosis of AILT was confirmed by histolopathologic features, immunohistochemical stains and molecular studies.Method: Specimens: Fresh groin excision lymph nodes. Flow cytometric analysis utilizing side scatter versus CD45. Case 1 (figure 1): The gated population consists of 10–12% B cells and 56–61% T cells. CD3, CD4 and CD5 stain 56–61% of the gated cells, while CD7 stains 41% of the gated cells, consistent with aberrant partial loss of CD7 by T cells. A population of abnormal T cells expresses CD10 with CD2, CD3, CD4 and CD7 but not with CD19 (B cells) consistent with aberrant CD10 coexpression by a subset of the T cells (12–19% of the gated cells). Staining for CD34 highlights less than 2% immature cells staining while CD64 stains less than 2% monocytic cells. Surface kappa and lambda staining does not show restriction (kappa: lambda 1–2:1). Cytoplasmic kappa and lambda staining is not interpretable due to non-specific antibody binding. Case 2 (figure 2): The gated population consists of 69% T- cells and 28–33% B- cells. A population of abnormal T cells expresses CD10 with CD2, CD3, CD4 and CD7 but not CD19 consistent with aberrant CD10 expression by a subpopulation of T cells (8–11% of gated cells). Intracellular B cell kappa lambda light chain restriction is not present and there is no aberrant co-expression of CD5, CD10 or CD43 by B cells. The diagnosis of AILT was confirmed by standard morphologic, immunoperoxidase and molecular methods subsequently in both of these cases.Discussion: Aberrant CD10 expression on neoplastic T cells has been shown with immunohistochemical staining of paraffin embedded tissue in AILT. Our cases show aberrant coexpression of CD10 by CD2, CD3, CD4 and CD7 positive T cells in more than 10% of gated cells by Flow cytometric analysis.Conclusion: Flow Cytometric analysis is a useful and reproducible tool for immunophenotyping AILT cells and should be considered for presumptive diagnosis of AILT. [Display omitted] [Display omitted]

Molecular Characterization of a Novel Subtype of Pediatric Acute Lymphoblastic Leukemia.

Gene expression profiling is a powerful tool to classify and predict subtypes of pediatric acute lymphoblastic leukemia (ALL). In addition to accurately classifying known subtypes of ALL, microarray analysis of 327 cases of ALL at our institution using Affymetrix U95 arrays identified a previously uncharacterized novel subgroup of 14 cases of B-ALL with a gene expression signature distinct from other leukemias, suggesting a unique pathogenesis. These cases lacked a recurring cytogenetic anomaly, showed a high frequency of aberrant expression of CD2, lacked molecular markers of other subtypes of ALL, and had a favorable prognosis. We have used several complementary candidate and genome-wide approaches to identify the molecular basis of this novel subtype. Gene expression profiling using Affymetrix U133A and B arrays was used to refine the signature, and when applied to an additional 36 cases of cytogenetically normal B-ALL, 5 cases were identified that share the novel gene expression profile. Intriguingly, the tyrosine kinase PDGFRA was overexpressed in the majority of the novel cases, with 6 novel cases showing exceedingly high levels of PDGFRA expression, which was confirmed by western blotting and flow cytometry. Unexpectedly, 3 of these cases harbored the FIP1L1-PDGFRA fusion characteristic of idiopathic hypereosinophilic syndrome. However, FISH examination demonstrated that this fusion was present in a minor subclone only. RT-PCR and FISH approaches failed to identify alternative PDGFRA fusions, and PDGFRA sequencing failed to identify evidence of activating mutations. Furthermore, in unstimulated cultures or following PDGF-AA stimulation, PDGFRA-overexpressing ALL blasts were not sensitive to imatinib (Gleevec). Other genes in the gene expression profile with potential roles in leukemogenesis, include include the B-cell receptor signaling mediator BRDG1 and the tyrosine phosphatase PTPRM. Using FISH and genomic sequencing of multiple candidates, no potentially leukemogenic abnormality has been identified. To determine if gene amplification, deletion, or copy-neutral loss of heterozygosity is characteristic of this subtype of ALL, genome-wide array-based comparative genomic hybridization and Affymetrix 100K single nucleotide polymorphism arrays were performed on all cases. Deletion of the CDKN2A/2B locus, and deletion of a region at 8p22–23.1 flanking the SPAG11 locus were each found in multiple cases, however no single unifying abnormality was identified. In summary, we have identified and characterized a novel subtype of B-ALL with unique gene expression signature, high incidence of CD2 and PDGFRA overexpression, and favorable outcome. Although no uniform causal molecular lesion has been identified, FIP1L1-PDGFRA fusion may represent one mechanism of PDGFRA overexpression. Further studies and analysis of additional cases may prove informative.

Aberrant expression of CD7, CD56, and CD79a antigens in acute myeloid leukemias

The prognostic significance of selected markers of leukemic cells is well known. CD7 and CD56 expression at diagnosis has been associated with low remission rates and biological aggressiveness in a significant proportion of acute leukemias. Among 46 patients with acute myeloid leukemia, we found CD7 expression in 15 cases (32.6%) and CD56 positivity in 10 patients (21.7%). Six of these myeloid leukemia cases (13%) showed expression of both CD7 and CD56. Four of 46 (8.7%) patients expressed CD79a. Among the 10 that were acute myeloblastic leukemia, 8 expressed CD7, 4 expressed CD56, and 4 were positive for CD79a. Thus, these markers were expressed early in hemopoietic ontogeny in the lesser-differentiated acute myeloid leukemia subtypes, including FAB M0, M1, and M2. Whereas CD7 and CD56 were each positive in 4 cases of acute myelomonocytic leukemia (FAB M4 subtype), there was no CD79a expression in the M4 cases. CD7 is expressed by mature T cells, NK cells, and an immature myeloid cell subset. NK cells and a T cell subset express CD56. By contrast, CD79a is a B cell marker that is assigned a high score of 2.0 in the differentiation of acute leukemias of ambiguous lineage in the WHO classification. The aberrant expression of CD7, CD56, and CD79a, representing the capacity of these leukemias for trilineal expression of leukocyte differentiation antigens, portends a poor prognosis.

Relative Contributions of Enzyme Cytochemistry and Flow Cytometric Immunophenotyping to the Evaluation of Acute Myeloid Leukemias With a Monocytic Component and of Flow Cytometric Immunophenotyping to the Evaluation of Absolute Monocytoses

We evaluated the contributions of enzyme cytochemical stains and flow cytometric immunophenotyping (FCI) data to detection of monocytic cells (MCs) in acute myelomonocytic and acute monocytic leukemias (AMMLs and AMoLs) and compared FCI findings in AMoL, chronic myelomonocytic leukemia (CMML), and normal peripheral blood (PB) and bone marrow (BM) monocytes to classify and evaluate absolute monocytoses (AMs). We reviewed 10 AMMLs and 6 AMoLs with a-naphthyl-acetate esterase (ANAE) and a-naphthyl-butyrate esterase stains and a complete FCI profile and compared FCI data for 6 AMoLs, 7 CMMLs, 2 AMs, and normal monocytes. We confirmed increased sensitivity of ANAE staining to FCI data in detecting MCs in AMML and AMoL. CD14 was insensitive for confirming MCs; other characteristic markers of MCs were absent or partially lost in AMML and AMoL. Aberrant expression of CD56 (detected in 50% of AMMLs and AMoLs), CD34, and CD117 indicated malignancy. The mature MCs of the CMMLs revealed variable FCI abnormalities (partial loss of CD13, CD14, and CD15; expression of CD56), as in the monoblasts of AMoL. These FCI abnormalities in morphologically mature MCs might indicate markers for CMML. AMs revealed FCI abnormalities, indicating clues to their correct classification as CMML.

Relative Contributions of Enzyme Cytochemistry and Flow Cytometric Immunophenotyping to the Evaluation of Acute Myeloid Leukemias with a Monocytic Component and of Flow Cytometric Immunophenotyping to the Evaluation of Absolute Monocytoses

We evaluated the contributions of enzyme cytochemical stains and flow cytometric immunophenotyping (FCI) data to detection of monocytic cells (MCs) in acute myelomonocytic and acute monocytic leukemias (AMMLs and AMoLs) and compared FCI findings in AMoL, chronic myelomonocytic leukemia (CMML), and normal peripheral blood (PB) and bone marrow (BM) monocytes to classify and evaluate absolute monocytoses (AMs). We reviewed 10 AMMLs and 6 AMoLs with α-naphthyl-acetate esterase (ANAE) and α-naphthyl-butyrate esterase stains and a complete FCI profile and compared FCI data for 6 AMoLs, 7 CMMLs, 2 AMs, and normal monocytes. We confirmed increased sensitivity of ANAE staining to FCI data in detecting MCs in AMML and AMoL. CD14 was insensitive for confirming MCs; other characteristic markers of MCs were absent or partially lost in AMML and AMoL. Aberrant expression of CD56 (detected in 50% of AMMLs and AMoLs), CD34, and CD117 indicated malignancy. The mature MCs of the CMMLs revealed variable FCI abnormalities (partial loss of CD13, CD14, and CD15; expression of CD56), as in the monoblasts of AMoL. These FCI abnormalities in morphologically mature MCs might indicate markers for CMML. AMs revealed FCI abnormalities, indicating clues to their correct classification as CMML.

Phenotypic Aberrations of Myeloid CD34+ Cells in Patients with Myelodysplastic Syndrome (MDS): Clinical Significance and Correlation with Cytogenetics Abnormalities.

Expression of abnormal markers in myeloid CD34+ cells of patients with MDS is common, but few immunophenotypic data have been compiled so far. Based on the statement that CD7 and TdT are markers associated with a bad prognosis in acute myeloid leukemia, we intended to describe the incidence of their aberrant expression in CD34+ cells and its role helping to establish the diagnosis of MDS.OBJECTIVES: To explore the aberrant expression of TdT and CD7 in myeloid CD34+ cells of MDS patients and to describe their possible correlation with cytogenetic features.DESIGN AND METHODS: Bone marrow specimens from 45 patients with MDS were included in this study (17 RA, 12 RARS, 5 CMML, 9 RAEB, 2 RAEB-t). In addition, we analyzed 28 samples of bone marrow from patients with cytopenias, but no diagnosis of MDS, as a cohort control. Immunophenotyping was performed with the following combination of monoclonal antibodies: TdT FITC / CD7 PE/ CD34 PCy-5. A case was regarded as positive for any of these markers when their expression was described in at least, 25% of myeloid CD34+ cells. Besides, adequate cytogenetic data and FISH analysis of chromosomes 5, 7 and 8 were obtained from 42 out of the 45 MDS samples.RESULTS: The percentage of CD34+ myeloid cells in MDS samples was fewer than 2% in 25 cases, ranged from 2–5% in 6 cases and was equal or greater than 5% in 14 cases. Aberrant expression of CD7 and/or TdT was observed in 28 cases: 20 were positive for CD7, 5 cases were positive for TdT and co-expression of both antigens was described in 3 cases. Prevalence of these abnormal markers was much higher in patients with MDS (28/45; 62%) than in the cohort control (2/28; 7%) . Besides, we identified at least one abnormal marker in 14 of the 16 patients with high risk MDS (9 RAEB, 5 CMML, 2 RAEB.t). According to the IPSS, karyotypes were divided into subgroups of favorable, intermediate or unfavorable, being classified 29 patients with favorable karyotypes and 13 patients with no favorable cytogenetics. In the first group, the abnormal expression of CD7 and/or TdT was detected in 13/29 patients (44.8%) and in 10/13 patients in the non-favourable group (76.9%).CONCLUSIONS: CD7 and/or TdT expression in myeloid CD34 + cells may be helpful in establishing the diagnosis of MDS. Prevalence of these aberrancies seems to be higher in cases with no favorable karyotypes, but a larger number of patients will be required to state significant differences. Possible correlation between high risk MDS patients and immunophenotypic aberrancies suggests the convenience of following the outcome of those low risk MDS patients who have expression of any of these abnormal markers.

Acute Megakaryoblastic Leukemia in Children without Down's Syndrome.

Acute megakaryoblastic leukemia (AMKL) in children without Down's syndrome occurs in less than 10% of childhood AML. Data on clinical presentation, immunological and genetic features and treatment strategies are rare.Between 6/1987 and 7/2003 89 children with AMKL (m=46; f=43) were treated according to the AML-BFM trials 87 (n=19), 93 (n=39) and 98 (n=31). AML FAB M7 was centrally confirmed by immunophenotyping (n=86) or immunohistology (n=3). The median age at diagnosis (1.5 yrs) was significantly lower compared to other children with AML (total group n=1,149; median age 7.8 yrs; p<0.004). The percentage of AMKL in the AML-BFM trials was 6%. Children with AMKL presented with anemia (93%), thrombocytopenia (88%) and hepatomegaly (83%). Hyperleukocytosis (> 100,000/μl) was less frequent (13%) compared to other AML subtypes (21%; p<0.0001). Immunophenotyping revealed the co-expression of stem cell antigens such as CD34 (61%) and CD117 (82%), myeloid antigens (CD33/CD13) and megakaryocytic antigens (CD41/CD42/CD61). The aberrant expression of CD7 (92%) was significantly increased (other AML subtypes 29%; p<0.0001). Cytogenetics revealed several aberrations such as t(1;22) (14%), monosomy 7 (12%) and trisomy 8 or 21 (7%), but mostly not isolated.The complete remission rate increased from 58% in study AML-BFM 87 to 77% and 83% in AML-BFM 93 and 98 (p<0.05), respectively. The outcome of all children with AMKL was as follows: 5-year event-free survival (EFS) 40±5%; disease-free survival (DFS) 49±6%; overall survival (OS) 42±6%. Compared to children with high risk (HR)-AML (n=714) treated in the AML-BFM studies, the EFS was similar (40±6% vs. 43±2%; p= 0.44). Due to poor response to relapse treatment, the OS tended to be inferior (AMKL 44±6% vs. HR-AML 52±2%; p=0.13).The treatment intensification in AML-BFM 93 and 98 with high-dose cytarabine and mitoxantrone (HAM) as second induction resulted in a significantly improved EFS (46±6%), DFS (58±7%) and OS (49±6%) compared to AML-BFM 87 (11±7%, 18 ±12%. 21±9%; p<0.009).There was no significant difference of outcome between children treated with chemotherapy only (n=54) or allogeneic stem cell transplantation (alloSCT) in 1st CR (n=13 ) DFS 50±7 vs 37±14; p=0.49; OS 54±7 vs 43±15%; p=0.47).Considering gender, age, WBC, blast percentage, immunophenotype or cytogenetic subgroup (available n=74; 83%) no prognostic factor could be identified. Children with poor response (n=19) or relapse (n=33) had a very unfavorable outcome. Only 5 of these children survived. Two children stayed alive in CR after relapse chemotherapy (follow-up 12.8 and 11.3 years), two after alloSCT (follow-up 1.4 and 2.4 years) and one child was alive with disease after alloSCT and relapse (follow-up 0.6 years). In conclusion, in children with AMKL intensive chemotherapy enabled a remission rate and a long-term survival comparable to other childhood HR-AML. AlloSCT seemed not to improve outcome. Therefore new approaches for the treatment of AMKL in children are needed.

Usefulness of CD10 Study by Multi-Colour Flow Cytometry in Angioimmunoblastic T-Cell Lymphoma.

Angioimmunoblastic T-cell lymphoma (AITL) is a distinct clinicopathological entity among peripheral T-cell lymphoma in the WHO classification. Whereas antigen “loss” or “deletion” of one or several pan-T cell antigens is a hepful feature of neoplastic lymphocytes in many T-cell lymphomas, no specific immunophenotypic patterns were available to recognize the tumour T-cells of AITL until recently. Indeed, Attygalle et al. reported that in this disorder neoplastic T-cells can be recognized by the aberrant expression of CD10 using immunochemistry in lymph nodes as well as in the involved extranodal sites. Lee et al. has also confirmed this specific phenotypic feature in cell suspension of lymph nodes using flow cytometry (FCM) in 3 cases of AITL. Here, we evaluated the CD10 expression by T cells in patients with AITL using four-colour FCM. The present study included lymph nodes (LN, n=10), peripheral blood (PB, n=5), bone marrow (n=1) and skin (n=1) samples from 13 patients with a diagnosis of AITL and with available cytologic histologic, immunologic and molecular data. Lymph nodes of reactive hyperplasia (n=13), B-cell lymphoma (n=23), other T-cell lymphoma (n=6) and peripheral blood from healthy donors (n=18) were used as control group. According with previous immunohistochemistry results, a fraction of T-cells expressed CD10 (using a level of at least 5% of all CD5+ cells) in 9/10 AITL lymph nodes with a mean number of 18%. Interestingly, among these 9 cases, 5 could be studied in peripheral blood also and all cases showed a fraction of T-cells expressing CD10, whatever be the lymphocytosis (median 1.1 109/l range 0.82 to 11.32 109/l). In three of these cases, tumoral T-cells presented also lack of surface CD3. In two cases of AITL diagnosed in LN, the aberrant CD10 expression by T-cells was found in bone marrow and skin, respectively. In the control group, T-cells were CD10 negative using the cut-off of 5%.In conclusion, we demonstrate that the assessment of CD10 expression by neoplastic T-cells can be achieved by multi-colour FCM in lymph nodes and involved extranodal sites. Our results are concordant with the statement of Attygalle that CD10 expression by T-cells can be used as a marker of both malignancy and AITL type. In addition, this is to our knowledge the first description of circulating CD10 neoplastic T-cells in AITL. Further study with a larger series of patients is required to confirm these data, to standardize the cut-off of positivity and to evaluate the sensibility of FCM versus immunohistochemistry. [Display omitted]

Mutation of CEBPA in Familial Acute Myeloid Leukaemia.

Two siblings (III-1 and III-5) were referred to St Bartholomew's Hospital for treatment for Acute Myeloid Leukaemia (AML) within the space of 2 weeks. There was a family history of AML (see figure) occurring in their father (II-3). All other family members were alive and well. Patient II-3 was diagnosed with AML in September 1963, aged 10, patient III-1 presented in February 2003, aged 30 and his sister Patient III-5, aged 18 the following week. A diagnosis of AML FAB type M2Eo was made for both III-1 and III-5 and cytogenetic evaluation was normal. Following the diagnosis, patients III-1 and III-5 received 4 courses of combination chemotherapy. Both achieved a complete remission after the first course of therapy and both presented soon after discharge following their final course of therapy with arthralgias, fever and malaise. At that time both patients had raised white counts (28.2 x 109/l and 20.4 x 109/l respectively) in the absence of overt sepsis and off G-CSF. The abnormalities in both patients resolved without medical intervention. At the time of writing, both patients are well with normal blood counts over 17 months from diagnosis. Patient III-1 was shown to have a del (C) mutation in CEBPA at nucleotide 212 in DNA extracted from a peripheral blood sample taken at diagnosis. The corresponding protein, S21fsX158, is predicted to terminate prematurely at codon 158. III-5 was also noted to have an identical del (C) mutation in her diagnostic sample. DNA extracted from their remission peripheral blood samples and germline DNA from buccal swabs, from all 3 affected individuals, further demonstrated the presence of the del(C) 212 mutation. In this family the CEBPA mutation appears to be fully penetrant: AML developed in all carriers of the 212 del(C) mutation, identified. The long latency period (10 years - II-3, 18 years - III-5 and 30 years - III-1) suggests that one or more additional mutations is necessary for the development of overt acute leukaemia. Mutation analysis was extended to include several other genes implicated in the development of AML (KRAS, NRAS, KIT, PTPN11, FLT3 and RUNX1), but no mutations were identified other than a second CEBPA mutation (duplication of nucleotides 1050 to 1085) on the other allele in patient III-1. Direct sequencing of CEBPA from DNA extracted from peripheral blood mononuclear cells from seven healthy family members (including II-1, II-2, III-2 and III-3) confirmed the presence of wild-type sequence in each case. In summary this study describes a family with familial CEBPA mutation associated with AML and having a putative regeneration syndrome following completion of therapy. All 3 affected individuals had been previously well with no prior medical history and no obvious dysmorphism. CEBPA mutation did not therefore appear to be part of a syndrome, such as FPD/AML seen with RUNX1 mutation. However, the resultant leukaemias did appear to be similar with myeloblastic morphology, normal cytogenetics, aberrant CD7 expression and the presence of Auer rods. This observation may enable other kindreds with similar phenotype to be selectably screened for CEBPA mutation. [Display omitted]

An Unusual Case of Leukemic Mantle Cell Lymphoma With a Blastoid Component Showing Loss of CD5 and Aberrant Expression of CD10

Characteristically, mantle cell lymphoma (MCL) expresses surface immunoglobulin (sIg), CD19, CD20, and CD5 and lacks CD10 and CD23. Rare CD5-MCL variants have been described. This report describes a case of leukemic MCL with morphologically and immunophenotypically distinct classic MCL and blastoid-variant MCL (BV-MCL) components. The classic MCL had typical morphologic features and immunophenotype (kappa sIg light chain-restricted and CD5+; CD10- and CD23-). The BV-MCL had larger nuclei and open chromatin; these cells also were kappa sIg light chain-restricted; however, they were CD10+ and CD5-. Fluorescence in situ hybridization studies demonstrated cyclin D1-immunoglobulin heavy chain gene fusion in both components; the bone marrow biopsy cellularity was replaced by CD10+ and cyclin D1+ and CD5-BV-MCL. This case illustrates the phenotypic heterogeneity of MCL and underscores the need for histopathologic correlation and, in some instances, ancillary genetic studies to accurately classify B-cell lymphomas.

Antibody binding capacity for evaluation of MDR-related proteins in acute promyelocytic leukemia: Onset versus relapse expression.

Multidrug resistance (MDR) remains a major obstacle for successful treatment in cancer, in particular in acute leukemia. In acute promyelocytic leukemia (APL), the high sensitivity to anthracyclines appears to be attributable to the low frequency of MDR proteins overexpression at onset even if 30% of patients still relapse and become resistant to therapy. In attempt to explain different blast cell sensitivity, we studied the expression of PGP, MRP1, MRP2, and LRP in 45 cases of APL, comparing onset of disease with relapse. PGP, LRP, and MRP on bone marrow or peripheral blood blast cells were evaluated by flow cytometry using the MRK-16, LRP-56, MRP-m6, and MRP2 antibodies and results expressed by the mean fluorescence index (MFI). The antibody binding capacity (ABC) for each MDR protein was also calculated. At diagnosis, only 2 of 45 patients overexpressed PGP and 1 overexpressed LRP. PGP and LRP overexpressing cases significantly grew up during disease progression and at second relapse mean PGP MFI and mean LRP MFI were significantly higher than at onset (P = 0.001 and P = 0.008, respectively). By analyzing ABC, the same trend was more evident because a significant increment of PGP and LRP was observed at second (P = 0.002 and P = 0.002, respectively), but even at first relapse (P = 0.018 and P = 0.002, respectively). No changes were demonstrated in MRP1 and MRP2 expression in any phase of disease considered. Our data confirm the low expression at diagnosis of proteins related to development of drug resistance in APL. The evidence of a relative easy induction of PGP and LRP, but not of MRP, can be useful in choosing drugs to employ for consolidation or rescue therapy.

Bone Marrow Mastocytosis Associated with IgM Kappa Plasma Cell Myeloma

An association between mastocytosis and monoclonal gammopathy is a relatively rare but well recognized clinical finding. In the majority of cases, however, overt myeloma or lymphoma is not detectable morphologically. Here we describe the case of a 51 year-old male patient first presenting with paresis of the right facial nerve and the serological finding of IgM kappa paraproteinemia. The patient did not have organomegaly, lytic bone lesions, or urticaria pigmentosa-type skin lesions. Histological examination of a trephine biopsy specimen revealed the unusual coexistence of plasma cell myeloma and mastocytosis. Immunohistochemically, plasma cells were found to exhibit a monotypic staining for Ig heavy chain mu and Ig light chain kappa, thus confirming their neoplastic nature. Mast cells showed prominent spindling and formed dense multifocal infiltrates, thus enabling the diagnosis of bone marrow mastocytosis. Immunohistochemically, mast cells expressed tryptase, chymase, and KIT (CD117). In addition, aberrant expression of CD25 on mast cells was detected, confirming the coexistence of a neoplastic mast cell-proliferative disorder. According to the WHO proposal for classification of hematopoietic malignancies, this unique case, showing the association of two very rare haematologic neoplasms, can therefore best be referred to as bone marrow mastocytosis associated with IgM kappa plasma cell myeloma (SM-AHNMD).

Immunophenotypic analysis of mast cells in mastocytosis: When and how to do it. Proposals of the Spanish Network on Mastocytosis (REMA).

Mastocytosis is a term used for a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow, liver, spleen, and lymph nodes, among others. In recent years, multiparameter flow cytometric studies have shown that pathologic MCs from patients with mastocytosis display unique aberrant immunophenotypic characteristics as compared with normal MCs. Among other features, pathologic MCs show aberrant expression of CD25 and CD2 antigens and abnormally high levels of the CD11c and CD35 complement receptors, the CD59 complement regulatory molecule, the CD63 lysosomal membrane antigen, and the CD69 early-activation antigen. In addition, MCs from mastocytosis express abnormally low levels of CD117 and unexpectedly high light scatter and autofluorescence characteristics. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this paper we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, information for their phenotypic characterization, and the criteria currently used for a correct interpretation of the immunophenotypic results obtained.

Analysis of CD10+ Hairy Cell Leukemia

Hairy cell leukemia (HCL) has been reported to sometimes express CD10. However, the reported frequencies have been quite variable and the significance of CD10 expression has not been addressed. Cases of HCL submitted to our flow cytometry service during a 2-year period were evaluated for CD10 expression. Information regarding demographics, clinical manifestations, tissue morphologic features, and response to treatment was reviewed. Of the 97 HCL cases identified, 10 expressed CD10. The level of CD10 staining was typically well above control levels and also could be detected easily by immunohistochemical analysis. All cases analyzed were negative for bcl-6. Our study suggests that approximately 10% of otherwise typical cases of HCL show aberrant CD10 expression. CD10+ HCL cases seem to be morphologically and clinically similar to CD10-HCL cases. Appreciating that HCL can express CD10 may be especially important when evaluating specimens with suboptimal morphologic features and/or limited immunophenotyping panels.

Analysis of CD10+ hairy cell leukemia.

Hairy cell leukemia (HCL) has been reported to sometimes express CD10. However, the reported frequencies have been quite variable and the significance of CD10 expression has not been addressed. Cases of HCL submitted to our flow cytometry service during a 2-year period were evaluated for CD10 expression. Information regarding demographics, clinical manifestations, tissue morphologic features, and response to treatment was reviewed. Of the 97 HCL cases identified, 10 expressed CD10. The level of CD10 staining was typically well above control levels and also could be detected easily by immunohistochemical analysis. All cases analyzed were negative for bcl-6. Our study suggests that approximately 10% of otherwise typical cases of HCL show aberrant CD10 expression. CD10+ HCL cases seem to be morphologically and clinically similar to CD10-HCL cases. Appreciating that HCL can express CD10 may be especially important when evaluating specimens with suboptimal morphologic features and/or limited immunophenotyping panels.

Evolution of Urticaria Pigmentosa into Indolent Systemic Mastocytosis: Abnormal Immunophenotype of Mast Cells without Evidence of c-kit Mutation ASP-816-VAL

Mastocytosis comprises a heterogeneous group of hematological disorders which are morphologically defined by proliferation and accumulation of tissue mast cells in one or more organs. Clinical manifestations of mastocytosis range from disseminated maculopapular skin lesions (=urticaria pigmentosa [UP]) that may spontaneously regress to highly aggressive neoplasms like mast cell leukemia or mast cell sarcoma. Recently, it could be shown that systemic mastocytosis (SM) is a clonal disorder often exhibiting mutations of c-kit, a protooncogene encoding the tyrosine kinase receptor for stem cell factor (SCF). Mutations of c-kit are considered to play a key role in the pathogenesis of mastocytosis. Therefore, we investigated the unique case of a 36 year-old male patient with indolent systemic mastocytosis (ISM) evolving from UP (cutaneous mastocytosis) by means of histology, immunophenotyping and molecular biology. At the time of initial diagnosis the bone marrow showed only a mild diffuse increase in mast cells but compact infiltrates were missing. The serum tryptase levels were normal. Five years later, however, the bone marrow histology displayed patchy compact mast cell infiltrates, which now allowed to establish the diagnosis of an ISM. The serum tryptase levels at this time were markedly elevated. At both time points, mast cells were analyzed by immunohistochemistry using anti-tryptase antibody AA1, by flow cytometry using antibodies against CD2 and CD25, and nested polymerase chain reaction (PCR) on laser-microdissected, single pooled mast cells. Immunohistochemistry revealed strong tryptase-positivity of mast cells in both cutaneous and bone marrow infiltrates. Flow cytometry yielded an aberrant expression of CD2 and CD25 on bone marrow mast cells. However, repeated thorough PCR analysis failed to unveil c-kit mutation in atypical mast cells of skin and bone marrow samples of both dates. These findings clearly show that ISM can evolve from UP. Moreover, our study provides further evidence that the c-kit mutation Asp-816-Val is not invariably present in ISM.