Aberrant Expression Of CD56 Research Articles

Peripheral T-Cell Lymphoma With a Natural Killer Cell/Cytotoxic T-Cell Phenotype and Aberrant Expression of CD19: A Case Report and Review of the Literature

Peripheral T-Cell Lymphoma With a Natural Killer Cell/Cytotoxic T-Cell Phenotype and Aberrant Expression of CD19: A Case Report and Review of the Literature

FLT3/ITD Associated with an Immature Immunophenotype in PML-RARα Leukemia

Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARα fusion gene resulting from translocation t(15;17) (q22;q12). Internal tandem duplication (ITD) of the FLT3 gene has been observed in approximately 35% of APLs, and large-scale studies have identified the presence of ITD as an adverse prognostic factor for acute myeloblastic leukemia (AML) patients. Aberrant expressions of surface antigens, such as CD2, CD34, and CD56, have been found in APL, but the implications of this are not well understood. We investigated the incidence of the FLT3/ITD mutation and FLT3/D835 (I836) point mutation in 25 APL patients. Incidence ratios of FLT3/ITD, D835 (I836), and both FLT3/ITD and D835 (I836) were 36%, 36% and 8%, respectively. FLT3/ITD+ cases showed a predominance of the bcr3 isoform (P=0.008) and M3v morphology (P<0.001). We found that all FLT3/ITD+ cases expressed CD2 (9 of 9) more frequently than that of FLT3/ITD− (1 of 16) (P<0.001), while only one of the CD2+ cases (1 of 10, 10%) did not harbor FLT3/ITD, and all CD2+CD34+ cases (5 of 5, 100%) harbored FLT3/ITD. In addition, quantitative polymerase chain reaction analysis showed that FLT3 mRNA was more abundantly expressed in FLT3/ITD+ than that in FLT3/ITD− (P=0.025), while there was no difference between D835(I836) + and D835(I836)− with regards to aberrant surface-antigen expression, expression levels of FLT3 mRNA, M3v morphology, and the bcr3 isoform of PML-RARα mRNA. This study demonstrates that the presence of FLT3/ITD, but not D835 (I836), is closely related to aberrant CD2 expression and high expression levels of FLT3 mRNA. Our findings also suggest that FLT3/ITD as a secondary genetic event may block differentiation at the immature stage of APL.

Langerhans cell sarcoma with an aberrant cytoplasmic CD3 expression

Langerhans cell sarcoma is a rare and aggressive high grade hematopoietic neoplasm with a dismal prognosis. It has a unique morphological and immunotypic profile with a CD1a/ langerin/S100 + phenotype. T cell lineage markers except for CD4 in Langerhans cell sarcoma have not been documented previously. We report a case of 86 year-old male of Caucasian descent who presented with an enlarging right neck mass over 2 months with an underlying unknown cause of anemia. Computed tomography scan of the neck, chest and abdomen revealed generalized lymphadenopathy and mild splenomegaly suspicious for lymphoma. Diagnostic core biopsy performed on right neck mass revealed a possible T cell lymphoma with expression of T cell lineage specific marker CD3 but conclusive diagnosis could not be made due to insufficient core biopsy sample. Further excisional biopsy performed on a left inguinal node showed a hematopoietic neoplasm with features of Langerhans cell sarcoma with a focal cytoplasmic CD3 expression in 30-40% of the tumor cells. PCR for T cell receptor (TCR) gene rearrangement failed to demonstrate a clonal gene rearrangement in the tumor cells arguing against a T cell lineage transdifferentiation, suggesting an aberrant CD3 expression. To the best of our knowledge, this case represents the first report of Langerhans cell sarcoma with an aberrant cytoplasmic CD3 expression.Virtual slideshttp://www.diagnosticpathology.diagnomx.eu/vs/2065486371761991

Clinicopathologic Features of B-Cell Lineage Neoplasms With Aberrant Expression of CD3

CD3 expression by immunohistochemistry was historically considered restricted to T-lineage or NK-lineage neoplasms but recently has been reported in rare cases of mature B-cell neoplasms, frequently in association with Epstein-Barr virus. Here, we describe the pathologic features of 21 B-cell lineage neoplasms that express CD3 protein by immunohistochemistry: 12 diffuse large B-cell lymphomas (DLBCLs); 2 plasmablastic lymphomas (PBLs); 4 plasma cell neoplasms; 2 Burkitt lymphomas; and 1 nodal follicular lymphoma, grade 3A. CD20 expression was negative or only partially positive in 13/21 cases. Epstein-Barr virus was positive in 3/20 tested cases (2 PBLs and 1 DLBCL). All tested neoplasms (14/14) had clonal immunoglobulin gene rearrangements, and no clonal T-cell gene rearrangements were detected (0/14). The 12 DLBCLs segregated into 2 main groups: 7 demonstrated features of plasmacytic differentiation but did not meet criteria for PBL, and 5 had anaplastic features. In addition to morphology, other features shared among the DLBCLs with plasmacytic differentiation, the plasma cell neoplasms, and the PBLs included extranodal presentation, cytoplasmic localization of CD3, and lack of expression of other T-cell antigens in most cases. In contrast, DLBCLs with anaplastic features and the single follicular lymphoma coexpressed multiple T-cell antigens in a predominantly membranous pattern and presented with nodal disease in a relatively younger patient population. Our data expand the spectrum of morphologic, phenotypic, and clinical features of B-cell neoplasms aberrantly expressing CD3. As these neoplasms often lack typical expression of B-cell antigens, knowledge of these features will help avoid misclassification.

Extracavitary KSHV-associated Large B-Cell Lymphoma

Primary effusion lymphoma (PEL) is a rare form of aggressive B-cell lymphoma in HIV patients, which typically presents with lymphomatous effusions in the body cavities without forming mass lesions. PEL is associated with Kaposi sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus-8) with distinct clinical and pathologic features. Rare cases of KSHV-associated large B-cell lymphoma (KSHV-LBL) have been observed in the lymph nodes or extranodal sites without lymphomatous effusions during the course of disease. KSHV-LBL is generally similar to classic PEL on the basis of the clinical presentation (HIV(+) male), morphology (immunoblastic, plasmablastic, or anaplastic), immunophenotype (CD45(+), CD20(-), CD79a(-), CD30(+), CD138(+), and EMA(+)), presence of Epstein-Barr virus infection, and clonal immunoglobulin gene rearrangements. However, it is not clear whether KSHV-LBL is a distinct entity or represents part of the spectrum of classic PEL; in particular, there is no consensus diagnostic term for KSHV-LBL. In this study, we investigated the clinicopathologic features of 9 cases of KSHV-LBL from our files. An additional 43 such cases and 84 cases of classic PEL from the English literature were reviewed and compared with each other. In contrast to the classic PEL, KSHV-LBL had a very significant lower expression of CD45 (74% vs. 94%, P=0.004) but significant higher expression of CD20 (17% vs. 5%, P=0.04) and CD138 (70% vs. 38%, P=0.05). KSHV-LBL also had slightly higher positivity of CD79a (23% vs. 5%, P=0.13) and immunoglobulin light chain expression, although the difference was not statistically significant [κ chain (12% vs. 0%) and λ chain (31% vs. 21%)]. The expressions of EMA and CD30 were slightly lower in KSHV-LBL compared with those observed in PEL (57% vs. 75% and 63% vs. 76%, respectively). Interestingly, 29% (10/34) of cases of KSHV-LBL revealed aberrant CD3 expression, which may mislead to a diagnosis of T-cell lymphoma, particularly anaplastic large cell lymphoma in combination with the anaplastic morphology and expression of CD30 and EMA. Although KSHV-LBL shows different clinical presentations and some variations in immunophenotype from classic PEL, it is still uncertain, on the basis of our findings, whether it is justifiable to separate them as 2 distinct entities. Nevertheless, we feel it is necessary to have a consensus diagnostic term, and we recommend a tentative one as "KSHV-associated large B-cell lymphoma (KSHV-LBL)" to replace many different names previously used.

Mast cell-related disorders presenting with Kounis syndrome

Systemic mastocytosis (SM) is a clinically heterogeneous group of disorders characterized by the accumulation of clonal MC in bone marrow (BM), and frequently also in other organs including the skin and/or the gastrointestinal tract. Currently, the clonal nature of the disease can be clearly established in most SM patients through the demonstration of somatic activating mutations involving the tyrosin-kinase regulatory domain of the Kit receptor [ [1] Garcia-Montero A.C. Jara-Acevedo M. Teodosio C. et al. KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients. Blood. 2006; 108: 2366-2372 Crossref PubMed Scopus (385) Google Scholar ]. In most cases, such KIT mutations lead to continuously ligand-independent autophosphorylation of the receptor, and initiation of downstream signalling events resulting in the release of MC-mediators. In recent years, detailed clinical, biological and molecular characterization of MC activation syndromes (MCAS) not fulfilling enough criteria for SM has been made [ [2] Alvarez-Twose I. Gonzalez de Olano D. Sanchez-Munoz L. et al. Clinical, biological, and molecular characteristics of clonal mast cell disorders presenting with systemic mast cell activation symptoms. J Allergy Clin Immunol. 2010; 125: 1269-1278 Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar ]. Based on the presence or absence of KIT mutations, MCAS are currently classified into clonal MCAS (c-MCAS) and non-clonal MCAS (nc-MCAS). Unlike BM MC from SM patients, in which aberrant CD25 expression and cytomorphological abnormalities, together with KIT mutations, are usually detected [ [3] Morgado J.M. Sanchez-Munoz L. Teodosio C.G. et al. Immunophenotyping in systemic mastocytosis diagnosis: ‘CD25 positive’ alone is more informative than the ‘CD25 and/or CD2’ WHO criterion. Mod Pathol. 2012; 25: 516-521 Crossref PubMed Scopus (54) Google Scholar ], BM MC from both c-MCAS and nc-MCAS cases frequently show a normal morphological appearance and/or immunophenotype [ [4] Valent P. Akin C. Arock M. et al. Definitions, criteria and global classification of mast cell disorders with special reference to mast cell activation syndromes: a consensus proposal. Int Arch Allergy Immunol. 2011; 157: 215-225 Crossref PubMed Scopus (417) Google Scholar ]. Patients with SM and MCAS suffer from systemic, usually recurrent symptoms, which are attributable to the release of MC mediators; Noteworthy, vasoconstriction-related features, such as increase of blood pressure, can be occasionally observed in a few patients during acute MC activation episodes [ [5] Brockow K. Jofer C. Behrendt H. Ring J. Anaphylaxis in patients with mastocytosis: a study on history, clinical features and risk factors in 120 patients. Allergy. 2008; 63: 226-232 Crossref PubMed Scopus (375) Google Scholar ].

Biological markers of cisplatin resistance in advanced testicular germ cell tumours

Germ cell tumours (GCTs) of the testis show exquisite sensitivity to treatment with cisplatin. Despite the high cure rates provided by platinum-based chemotherapy, 10-20% of patients die from progressive disease. Although various cellular pathways may influence cisplatin efficacy, their actual impact has not been comprehensively investigated in advanced GCTs. The objective of the present study was to clarify the role of the expression status of proteins involved in the Rb and p53 tumour suppressor pathways in sensitivity and resistance of GCTs to cisplatin-based chemotherapy. Paraffin-embedded tumour tissues from 84 patients with advanced GCT treated with cisplatin-based chemotherapy were analysed. Immunohistochemical expression of proteins p53 and mdm2, and the G1-phase cyclins D1 and D2 (CD1 and CD2) was assessed and correlated with the clinical course. The percentages of positive expression of p53, mdm2, CD1 and CD2 were 56, 57, 37.5 and 55%, respectively. From univariate analysis, there was no significant association between p53, mdm2 or CD1 expression and outcome. Instead, positive CD2 expression was found to be marginally associated with shorter median duration of progression-free survival (PFS) (p=0.06). In multivariate analysis, none of the molecular markers retained statistical significance with treatment response or survival. Tissular expression of p53, mdm2 and CD1 is not associated with prognosis or treatment response in patients with advanced GCT. Aberrant CD2 expression appears to further determine a shorter PFS. Larger and further studies are required to validate CD2 as a marker of cisplatin resistance.

Current state of biology and diagnosis of clonal mast cell diseases in adults

Mastocytosis comprises a heterogeneous group of disorders characterized by the presence of clonal mast cells (MC) in organs such as skin, bone marrow (BM), and gastrointestinal tract, among other tissues. The clonal nature of the disease can be established in most adult patients by the demonstration of activating KIT mutations in their BM MC. When highly sensitive techniques capable of identifying cells present at very low frequencies in a sample are applied, BM MC from virtually all systemic mastocytosis patients display unique immunophenotypical features, particularly the aberrant expression of CD25. By contrast, large, multifocal BM MC aggregates (the only World Health Organization major criterion for systemic mastocytosis) are absent in a significant proportion of patients fulfilling at least three minor criteria for systemic mastocytosis, particularly in subjects studied at early stages of the disease with very low MC burden. Moreover, recent molecular and immunophenotypical investigations of BM MC from patients with indolent systemic mastocytosis have revealed a close association of some biological features (e.g., multilineage involvement of hematopoiesis by the KIT mutation and an immature mast cell immunophenotype) with an increased risk for disease progression. These observations support the fact that, although the current consensus diagnostic criteria for systemic mastocytosis have been a major advance for the diagnosis and classification of the disease, rationale usage of the most sensitive diagnostic techniques available nowadays is needed to improve the diagnosis, refine the classification, and reach objective prognostic stratification of adult mastocytosis.

Immunophenotyping in systemic mastocytosis diagnosis: ‘CD25 positive' alone is more informative than the ‘CD25 and/or CD2' WHO criterion

Aberrant expression of CD2 and/or CD25 by bone marrow, peripheral blood or other extracutaneous tissue mast cells is currently used as a minor World Health Organization diagnostic criterion for systemic mastocytosis. However, the diagnostic utility of CD2 versus CD25 expression by mast cells has not been prospectively evaluated in a large series of systemic mastocytosis. Here we evaluate the sensitivity and specificity of CD2 versus CD25 expression in the diagnosis of systemic mastocytosis. Mast cells from a total of 886 bone marrow and 153 other non-bone marrow extracutaneous tissue samples were analysed by multiparameter flow cytometry following the guidelines of the Spanish Network on Mastocytosis at two different laboratories. The ‘CD25+ and/or CD2+ bone marrow mast cells' World Health Organization criterion showed an overall sensitivity of 100% with 99.0% specificity for the diagnosis of systemic mastocytosis whereas CD25 expression alone presented a similar sensitivity (100%) with a slightly higher specificity (99.2%). Inclusion of CD2 did not improve the sensitivity of the test and it decreased its specificity. In tissues other than bone marrow, the mast cell phenotypic criterion revealed to be less sensitive. In summary, CD2 expression does not contribute to improve the diagnosis of systemic mastocytosis when compared with aberrant CD25 expression alone, which supports the need to update and replace the minor World Health Organization ‘CD25+ and/or CD2+' mast cell phenotypic diagnostic criterion by a major criterion based exclusively on CD25 expression.

Clinicopathological features of hepatosplenic T cell lymphoma: a single centre experience from India

In a first series from India, we report 9 cases of hepatosplenic T cell lymphoma (HSTCL) seen in 23 months accounting for 4.2% of all mature T-non-Hodgkin lymphomas (NHLs) in our institution. All patients presented with organomegaly, cytopenias and had evidence of bone marrow involvement. The tumor cells had a blastic (55%) morphology with predominantly intrasinusoidal (33.3%) or intrasinusoidal with an additional interstitial component (33.3%). On flow cytometry, the classical phenotype (CD3+, CD7+, CD4−, CD8−, CD5−, CD56+/−) was seen only in 4 patients. Unusual variations included CD45 (overexpression), CD7 (dim expression), CD3 (overexpression, heterogeneous and dim), CD2 (overexpression), CD5 (heterogeneous), CD8 (heterogeneous or dim or overexpression) and aberrant expression of CD19. Fluoresvent in situ hybridisation (FISH) and karyotyping was abnormal in 5 out of 7 patients evaluated. All of the 5 cases showed abnormalities in chromosome 7 (ring chromosome or isochromosome 7q). Five patients died of disease and related complications in a span of 1–3 months after diagnosis whereas 4 were alive at their last follow up out of which 2 had documented a relapse. In our series, HSTCL was characterized by typical clinical and variable immunophenotypic features and a dismal clinical outcome.

A NUP98-NSD1 Fusion Gene Can Be Detected in a Small Subset of Adult AML Patients with Normal Karyotype

Abstract 2531 Introduction:Fusion genes can be detected in approximately 30–35% of all AML cases and usually are the result of a cytogenetically detectable chromosomal rearrangement. Very recently, a novel fusion gene has been described in AML with normal karyotype (Hollink et al, Blood, 2011). This cryptic fusion involves nucleophosmin 98kD (NUP98) in chromosomal band 11p15 and the non homeobox gene NSD1 in chromosomal band 5q35. NUP98-NSD1 has been described in this single study with a frequency of 16.1% in pediatric and 2.3% in adult AML patients with distinct characteristics (e.g. mutual exclusivity with NPM1) and dismal prognosis. Aim: The aim of this study was to further evaluate NUP98-NSD1 rearrangements in adult AML with normal karyotype (NK) for frequency, association with other mutations and impact on outcome. Patients and Methods: Screening for NUP98-NSD1 fusion gene was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) in a total cohort of 148 de novo AML patients with NK and NPM1 wildtype status. The NUP98-NSD1 positive cases were verified by direct Sanger Sequencing of the PCR products. The cohort was composed of 84 males and 64 females. Median age was 55.4 years (range: 15.7 to 85.8 years). Further mutation analysis was available in subcohorts: FLT3-ITD (n=32 mut/117 screened), CEBPA (n=22 mut/124 screened), MLL-PTD (n=32 mut/117 screened) and RUNX1 (n=26 mut/83 screened). Results: In total, in 8/148 (5.4%) patients a NUP98-NDS1 fusion transcript was detected. NUP98-NDS1-positive cases had significantly higher platelet counts (median 221 vs 87 × 10e9/L; p=0.001). Patients with NUP98-NDS1 were younger than the NUP98-NDS1-negative patients (median: 43.5 years vs 55.4 years, p=0.067). Sex (5 male vs. 3 female), white blood cell count and hemoglobin levels at diagnosis were not different compared to NUP98-NDS1-negative cases. Cytomorphology revealed AML with minimal differential differentiation (n=4), with maturation (n=1), and myelomonocytic AML (n=3). In 3 NUP98-NDS1-positive cases immunophenotyping data was available and all 3 cases aberrantly expressed CD7. NUP98-NDS1-positive cases have a higher frequency of FLT3-ITD compared to NUP98-NDS1-negative cases (5/8, 62.5% vs. 27/140, 19.3%; p=0.015) and were mutually exclusive of CEBPA and RUNX1 mutations. With respect to survival the NUP98-NDS1-positive cases had a worse event free survival compared to NUP98-NDS1-negative cases (median 5.1 months vs. 25.2 months; p=0.054). Conclusions: A NUP98-NSD1 fusion transcript was detected in 5.4% of normal karyotype adult AML patients without NPM1 mutation. NUP98-NSD1-positive cases are characterized by younger age, high coincidence of FLT3-ITD, aberrant expression of CD7, relatively high platelet counts, and a short event free survival. Thus NUP98-NSD1 translocations seem to define a new subgroup of NK-AML. Importantly, in this prognostically adverse and so far cytogenetically undetectable group close and sensitive PCR based monitoring for minimal residual disease is available. Thus, this data suggests to perform PCR based screening for NUP98-NSD1 in AML with normal karyotype that lack NPM1 and CEBPA mutations. Disclosures:Fasan:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Immunophenotyping Features in Acute Myeloid Leukemia (AML) with NPM1+ and/or FLT3+

Abstract 4908Immunophenotyping Features in Acute Myeloid Leukemia (AML) with NPM1 and/or FLT-3 PositivePervin Topçuoglu, Klara Dalva, Sinem Civriz Bozdag, Önder Arslan, Muhit Özcan, Osman Ýlhan, Hamdi Akan, Meral Beksaç, Günhan Gürman Aim:We aimed to evaluate immunophenotypical (IP) features in AML pts with NPM1+ and/or FLT3+ except on acute promyelocytic leukemia. Patients&Method:Between Nov 2009 and Feb 2011, we retrospectively analyzed IP features by flow cytometry (FCM) in 51 pts (46M;17F) with new diagnosed AML. Median age was 46 years (range: 14–71 ys). The mutations of NMP1 and FLT-3 TKD&ITD were determined by the methods of RQ-PCR or RFLP, respectively in the samples of bone marrow (n=31) or periheral blood (n=20) at the diagnosis. Antigenic expression of leukemic cells was analyzed by four-color FCM (FITC, PE, PerCP&APC) based by Nomdedeu et al researh (Leuk res 2011; 35:163) (Table-1). Results:We detected NMP1+ mutation in 16 patients. Of these, three were associated with mutations of FLT3-ITD (n=2) or -TKD (n=1). Twelve patients had FLT3+ (9 ITD or 3 TKD). More than half of the patients without any mutation were CD15+/CD34+/HLA-DR+ and 11.5% for CD34 negative. Similarly, the patients with FLT-3 positive were mostly CD34+ as the pts w/o any mutations. Contrary, most of the pts with NMP1+ were CD34 negative (56.3%) (Table 1). When evaluated the complete IP in leukemic cells, the expression of CD123 was significantly marked in the patients with NPM1+ and/or FLT3+ than those w/o mutations (p=0.008). While the co-expression of CD7 and CD117 was found in 67% of the pts w/o any mutations, 30% of the pts with NMP1 and/or FLT-3 ITD (p=0.01). CD56 expression was detected in more pts with NMP1+ than those with FLT-3+ (40% vs 8%, p=0.04). Besides, CD36 expression was positive in the all pts with FLT3-ITD than TKD+ (p=0.005). More intensive CD33 expression was seen in NMP1+ pts. The expression of CD64 was similar in all three mutations. Conclusion:Though NMP1 mutation was associated more CD34+ cells, more FLT3+ pts had CD34 positivity. The expression of CD123 was especially associated with the mutations. Aberrant expression of CD56 was in more pts with NPM1+, but CD36 for FLT3-ITD. These data might be a step for a study aiming to show a correlation between the type of mutations combined with IP features of leukemic cells and clinical characteristics or disease course of AML pts.Table 1:Mutation and Immunophenotypical features in AMLType 1 CD15-/ CD34-/ HLA-DR-n(%)Type 2 CD15-/ CD34-/ HLA-DR+ n(%)Type 3 CD15-/ CD34+/ HLA-DR+ n(%)Type 4 CD15+/ CD34+/ HLA-DR+ n(%)Type 5 CD15+/ CD34-/ HLA-DR+ n(%)Type 6 CD15+/ CD34-/ HLA-DR- n(%)Type 7 CD15-/ CD34+/ HLA-DR- n(%)Type 8 CD15+/ CD34+/ HLA-DR- n(%)NPM + (n=16)2 (12,5)2 (12,5)1 (6,3)4 (25)5 (31,3)1 (6,3)01 (6,3)FLT3 ITD + (n=9)002 (22,2)5 (55,5)1 (11,1)01 (11,1)0FLT3 TKD + (n=3)01 (33,3)01 (33,3)1 (33,3)000NPHFLT-3 + (n=3)01 (33,3)1 (33,3)01 (33,3)000 Disclosures:No relevant conflicts of interest to declare.

Myelodysplasia-Associated Immunophenotypic Abnormalities of Bone Marrow (BM) Cells in Multiple Myeloma (MM): Are They Present At Diagnosis or Can Be Induced by Lenalidomide?

Abstract 5066Although secondary primary malignancies such as myelodysplastic syndromes (MDS) have been mostly associated with alkylating therapies in MM, a potential association with lenalidomide has also been recently reported. However, because of the high median age of MM patients, it could be hypothesized that dysplastic features may be already present in MM at diagnosis, but this remains largely unexplored.Here, we used multiparameter flow cytometry (MFC) for detailed analysis of the distribution and immunophenotypic patterns of maturation of different BM hematopoietic cell compartments at diagnosis (baseline) in 33 MM patients (15 symptomatic and 18 high-risk smoldering MM; SMM). In addition, we analyzed the BM of 22 high-risk SMM patients after 9 courses of lenalidomide plus dexamethasone (LenDex), 8 of whom were from the baseline cohort (these patients were included in the QUIREDEX trial and received an induction phase of 9 four-week cycles of LenDex followed by maintenance with lenalidomide until disease progression).Overall, multiple phenotypic abnormalities (>1 phenotypic abnormality in ≥1 cell lineages) were found at diagnosis in 4 of 15 (27%) symptomatic MM and 2 of 18 (11%) SMM patients, one of the latter two SMM cases also displaying morphological abnormalities consistent with MDS. In turn, a similar (p>.05) frequency of isolated phenotypic abnormalities (only 1 phenotypic abnormality in 1 lineage -unilineage-, or >1 lineages -multilineage-) was noted at baseline in symptomatic MM (6 of 15; 40%) and high-risk SMM (9 of 18; 50%) cases. Despite this, an imbalance (p=.03) in favor of isolated multilineage vs unilineage abnormalities was observed in symptomatic (67% vs 33%) compared to smoldering cases (22% vs 78%). Particularly, the frequency of phenotypically-altered monocytic cells was significantly higher in symptomatic vs. smoldering MM (80% vs 27%; p=.03), mainly due to aberrant CD56 expression (60% vs 9%; p=.02). Noteworthy, phenotypic abnormalities involving other minor BM cell compartments (e.g. basophils and eosinophils) were restricted to symptomatic MM.Due to recent concerns about the potential risk for secondary MDS following lenalidomide treatment, we also analyzed the relative distribution and phenotypic patterns of the different BM cell compartments in SMM patients after 9 courses of LenDex. Except for a higher frequency of phenotypically altered erythroblasts (23% vs 0%; p=.03), no significant differences were noted between SMM patients after treatment vs the baseline cohort, concerning the frequency, extent and type of the phenotypic abnormalities detected. In order to further assess whether lenalidomide could either revert the phenotypic abnormalities present in SMM at baseline, or induce more abnormalities, we specifically focused our analysis in those 8 cases with paired studies performed at diagnosis and after 9 cycles of LenDex. Among these cases, three had no abnormalities at diagnosis: one remained without abnormalities after therapy while the two other developed isolated (unilineage and multilineage) abnormalities. Four cases presented isolated unilineage alterations at baseline; after induction one of these four patients reverted to a normal phenotype, two cases showed similar isolated unilineage alterations, and one patient developed multiple abnormalities. Similarly, one patient with isolated multilineage abnormalities at diagnosis, showed the same pattern after therapy.Our results suggest that a significant proportion of MM and SMM patients already show altered MFC phenotypic features in different BM hematopoietic cell compartments at diagnosis, and do not support a clear association between LenDex therapy and MDS. However, whether or not the altered cells harbor a higher susceptibility for further multistep accumulation of genetic defects remains to be elucidated. Disclosures:Off Label Use: lenalidomide is not approved for smoldering myeloma. Paiva:Celgene: Honoraria; Janssen: Honoraria. Mateos:Celgene: Honoraria; Janssen: Honoraria. Lahuerta:Celgene: Honoraria; Janssen: Honoraria. Rosiñol:Celgene: Honoraria; Janssen: Honoraria. Blade:Celgene: Honoraria; Janssen: Honoraria. San Miguel:Celgene: Honoraria; Janssen-Cilag: Honoraria.

POTENTIAL ROLE of 8-COLOR FLOW CYTOMETRY for CHARACTERIZATION of MDS with Del(5q) ABNORMALITIES

Abstract 5020Four-colour based flow cytometry (FCM) of bone marrow (BM) samples has become a valuable tool in the diagnostics of patients (pts) with myelodysplastic syndrome (MDS). It is still unknown, whether aberrations detected by FCM differ between certain cytogenetic subgroups. By using an 8-color (5-tube) FCM diagnostic panel we aimed to investigate the antigen patterns of MDS pts with del(5q) abnormalities and compared these to MDS pts with non-del(5q) MDS.BM samples of healthy donors (n=19) and 57 MDS pts including 28 pts (15 RCMD; 3 RAEB-1; 8 RAEB-2; 1 RAEB-t; 1 CMMoL-1) with del(5q) MDS (single n=15) and 29 pts (1 RCUD; 2 RA±RS; 17 RCMD; 2 RAEB-2; 7 sAML including 1 RAEB-t) with non-del(5q) MDS were investigated. FCM procedure was performed at a FACS Canto II (200,000 events) and standardized including the analysis of all samples after overnight storage at room temperature and erythrocytes lysis prior to antibody staining. Thresholds were set according to median ± 2SD and/or ½ log differences compared to healthy BM. Aberrations were scored according to the FCSS by Wells et al. 2003 and modified by Van de Loosdrecht et al. 2008.In del(5q) MDS several phenotypic characteristics differed to non-del(5q) pts. Of note, lineage infidelity (CD2, CD5, CD7, CD19, CD56) was detectable in significantly fewer del(5q) pts compared to non-del(5q) MDS (in granulocytes: 8% vs. 33%, p=0.040 and in monocytes: 24% vs. 56%, p=0.027). Thus, especially aberrant CD56 expression contributed to these results with only small subpopulations being positive in del(5q) vs. often more than 50% of aberrant granulocytes/monocytes in non-del(5q) MDS pts. Additionally, the lymphoid-to-granulocytes/monocytes-ratios were higher in del(5q) (p=0.029 and 0.025) and hypogranularity was more pronounced in those pts (p=0.05). Intensity of expression of CD14 on monocytes as well as CD71 on granulocytes showed significant differences (2478 vs. 4402, p=0.035 and 2098 vs. 1552, p=0.007). Most of the above mentioned differences could also be confirmed if only RCMD pts of both groups were considered (e.g. lineage infidelity in granulocytes: 0% vs. 36%, p=0.041). Furthermore, trends were seen for a lower CD45-lymphocytes-to-blasts-ratio (4.8 vs. 5.4; p=0.09), higher CD33 intensity on blasts (2416 vs. 1673, p=0.08), and higher qualitative CD36 expression on granulocytes (3.2% vs. 2.5%, p=0.0619) as well as quantitative expression on monocytes (1940 vs. 663, p=0.06). Finally, monitoring of aberrations by FCM allowed for minimal residual disease monitoring in del(5q) MDS pts. Interestingly, the decreased CD19 expression on blasts seemed to be stable independent from the extent of treatment response.Eight-colour based FCM is a valuable tool in the diagnostics of MDS. It might also be able to distinguish del(5q) MDS from other MDS subtypes. Disclosures:No relevant conflicts of interest to declare.

An immature immunophenotype of bone marrow mast cells predicts for multilineage D816V KIT mutation in systemic mastocytosis

D816V KIT mutation of bone marrow (BM) mast cells (MC) is a common feature to systemic mastocytosis (SM) patients. Nevertheless, occurrence of the KIT mutation in BM cell compartments other than MC is associated with progression to more aggressive forms of the disease and poor outcome in indolent SM (ISM). Here, we assessed the potential association between the immunophenotype of MC and multilineage KIT mutation in the BM of SM patients through the investigation of the flow cytometric protein expression profile (PEP) of bone marrow mast cells (BMMC) from 70 control individuals and 206 SM patients, classified according to the WHO (World Health Organization), and the degree of involvement of BM hematopoiesis by the D816V KIT mutation; additionally, we developed a score-based class prediction algorithm for the detection of SM cases with multilineage mutation. Our results show that aberrant expression of CD25 with a FcɛRI(lo), FSC(lo), SSC(lo) and CD45(lo) immature phenotype of BMMC, in the absence of coexisting normal MC in the BM, was associated with multilineage involvement by the D816V KIT mutation, regardless of the diagnostic subtype of the disease (for example, indolent vs aggressive SM), which supports the utility of the immunophenotype of BMMC as a surrogate marker to screen for multilineage KIT mutation in ISM.

Type II Enteropathy-Associated T-Cell Lymphoma

Enteropathy-associated T-cell lymphoma (EATL), an uncommon lymphoma of intestinal intraepithelial T lymphocytes, occurs with a higher frequency in northern Europe due to association with celiac disease. Data on the occurrence of EATL in the Asian population, among whom celiac disease is very rare, are conflicting. This study aimed to characterize EATL encountered in the Chinese population in Hong Kong. Eighteen cases were identified, all fulfilling the criteria of type II rather than classical EATL. The patients, including 13 men and 5 women, had a median age of 62 years. Most presented with small bowel perforation, and there was no history of malabsorption. The clinical course was aggressive, with 14 of 16 patients dying of progressive disease or complications, usually within 1 year. The histologic features were practically identical in all cases. The central zone of the tumor showed ulceration with or without perforation and was characterized by monotonous transmural infiltration of the bowel by small-sized or medium-sized lymphoma cells with few admixed inflammatory cells and no coagulative necrosis. The peripheral zone featured lateral spread of lymphoma cells in the mucosa, accompanied by variable involvement of the submucosa and muscularis. In all cases, there was an intraepithelial lymphocytosis zone contiguous or discontinuous with the peripheral zone, which was characterized by infiltration of the intestinal epithelium by nonatypical small lymphocytes, and not accompanied by other histologic changes of enteropathy. The most common phenotype of the lymphoma cells was CD3+, CD5-, CD4-, CD8+, CD56+, TIA1+, CD30-, and Epstein-Barr virus, and 2 cases showed aberrant expression of CD20. A remarkable finding was that 14 (78%) cases expressed γδ T-cell receptor, and only 6 (33%) expressed αβ T-cell receptor (with 3 cases coexpressing both T-cell receptors and 1 case expressing neither). The immunophenotype of the intraepithelial lymphocytes was either discordant (particularly with respect to CD8 and CD56 expressions) or concordant with the lymphoma cells of the corresponding cases. Thus, this study shows that EATL occurring in the Chinese population is exclusively of type II. In contrast to several studies, intraepithelial lymphocytosis can be consistently demonstrated and this component seems to represent a precursor lesion of EATL rather than a manifestation of celiac disease. In view of the differences in epidemiology and clinicopathologic features, we believe it is justified to separate out type II EATL from the EATL category as a distinct form of lymphoma, for which we propose the designation "monomorphic intestinal T-cell lymphoma."

Cytogenetic analysis of acute myeloid leukemia with t(8;21) from a tertiary care center in India with correlation between clinicopathologic characteristics and molecular analysis

The t(8;21)(q22;q22) is the most common translocation in acute myeloid leukemia (AML). We describe the clinicopathologic and cytogenetic profile of 117 patients with t(8;21) AML. There were 76 males and 88 adults. The median age was 26 years. Most patients (80%) had AML M2. Dysplasia was present in 68% of patients and eosinophilia in 18%. Eight patients had fewer than 20% blasts. Additional chromosomal aberrations were seen in 103 patients (88%) with loss of a sex chromosome (LSC) in 78 patients (66%) and deletion 9q in 21 (18%). The other recurrent abnormalities were trisomies 4, 8 and 15, monosomy 17 and deletion 7q (less than 5% each). Three- or four-way variant t(8;21) were seen in 6% of patients and 3% had tetraploidy. Aberrant expression of CD19 was seen in 54% of patients. FLT3 mutations were seen in 7.5% of patients (3/40) and c-KIT mutations in 6.6% (2/30). None had NPM1 or JAK2 V617F mutations. One patient had a granulocytic sarcoma. Complete remission was achieved in 96% of the 26 newly diagnosed patients after first induction. The median follow-up was 25 months (range 4–68). The overall survival was 69% at 31 months.

Plasma Cell Leukemia: Case Series From a Tertiary Center with Review of Literature

Plasma cell leukemia is an unusual manifestation of multiple myeloma, reported to occur in 2% of newly diagnosed patients. It may either present at the time of diagnosis (primary) or evolve as a late feature in the course of multiple myeloma (secondary). Most clinical signs of myeloma are observed in plasma cell leukemia, although osteolytic lesions and bone pain are less frequent and lymphadenopathy, organomegaly and renal failure are more often present. The immunophenotype of plasma cell leukemia differs typically from that of myeloma by lack of aberrant CD56 expression. An abnormal karyotype is more frequently found in plasma cell leukemia and there is higher incidence of unfavourable cytogenetics. Plasma cell leukemia is an aggressive disease, characterized by a fulminant course and a short survival. We are reporting cases of this rare condition which presented at our center over 3years along with review of literature.

Case study interpretation—Houston: Case 1

Case study interpretation—Houston: Case 1

Prognostic significance of immunophenotypic and karyotypic features of Philadelphia positive B‐lymphoblastic leukemia in the era of tyrosine kinase inhibitors

Philadelphia chromosome (Ph)-positive B-lymphoblastic leukemia exhibits immunophenotypic, karyotypic, and molecular genetic heterogeneity. The prognostic significance of these parameters was assessed in the context of intensive tyrosine kinase inhibitor (TKI)-based chemotherapy. The authors studied 65 adult patients with Ph-positive acute lymphoblastic leukemia (ALL) who received treatment with TKI-based therapy, correlated their clinicopathologic heterogeneity with patient outcome, and compared the findings with those from 60 adult patients with diploid B-cell ALL who received similar chemotherapy without a TKI. Ph-positive ALL was associated with older age (P = .01), the common-B immunophenotype characterized by a greater frequency of CD13 (alanine aminopeptidase) coexpression (P = .004), CD66c (carcinoembryonic antigen-related cell adhesion molecule 3) expression (P = .007), and CD25 (interleukin-2 receptor alpha chain) expression (P < .001) and with a lower frequency of CD15 (3-fucosyl-N-acetyl-lactosamine) expression (P < .001). Conventional karyotypic analyses indicated that the Ph chromosome was the sole abnormality in 19 patients (30%), was present with other aberrancies in 43 patients (65%), and was absent (detectable only by fluorescence in situ hybridization [FISH] or quantitative reverse transcriptase-polymerase chain reaction [RT-PCR] analysis) in 3 patients (5%). The presence of the breakpoint cluster region-v-Abelson murine leukemia viral oncogene homolog fusion gene (BCR-ABL) was confirmed in all patients by FISH or RT-PCR (the 190-kDa protein [p190] construct was present in 49 patients [77%], and the p210 fusion transcript construct was present in 15 patients [23%]). The presence of a supernumerary Ph chromosome was correlated with a higher incidence of CD20 (B-lymphocyte antigen, nonglycosylated phosphoprotein) expression (P < .001), whereas the p210 construct was correlated with aberrant CD25 expression (P = .05). Outcomes were not influenced by the degree of karyotypic complexity (including the presence or absence of a supernumerary Ph chromosome), CD20 expression, or myeloid antigen expression (CD13, CD33 [myeloid lineage transmembrane receptor], CD66c). CD25 expression was associated with inferior survival in univariate analysis (P = .051) but not in multivariate analysis (P = .092). In the context of intensive, TKI-based chemotherapy, the immunophenotypic, karyotypic, and molecular heterogeneity of Ph-positive ALL no longer influences outcome.