Multicentric Analyses of the CD148, CD180, and CD200 Combination for the Diagnosis of Mature B-Cell Neoplasm Using Flow Cytometry

Abstract Introduction Diagnosis of mature B-cells proliferations, especially those involving the spleen, do not always falls into any of the WHO types of B-cell neoplasms using standart diagnosis tools. This situation in notably encountered in the case of the differential diagnosis of marginal zone lymphoma (MZL), atypical chronic lymphocytic leukemia (aCLL), mantle cell lymphoma (MCL), and lymphoplasmacytic lymphoma (LPL), mostly due to the lack of immunological positive markers. In order to find new markers to discriminate between these different malignancies, we have previously developed a proteomic strategy based on the analyses of plasma membrane microparticles and proposed two new specific markers: CD148 and CD1801,2 for MCL and MZL respectively. The simultaneous use of these two markers, together with the CD200 that is positive in most cases of CLL and negative in MCL could be of great interest to better assess the differential diagnosis. Methods Flow cytometry analyses have been realized in Nancy and Strasbourg hospitals by combining these three markers: CD148 (Clone 143-41 FITC); CD180 (Clone G28.8 PE) and CD200 (Clone OX104 APC). Expression profile of these proteins was established on a well characterized set of patients (N=287): CLL with a Matutes score > 3 (N=81); MCL harboring t(11;14) translocation or CCND1 overexpression (N=44); LPL (N=58) classified following cytological morphology, IgM peak and the positivity of CD38 and/or Myd88 mutation, MZL (N=84), displaying a CD5- CD23- immunophenotype associated to a splenomegaly and 20 controls. For each group the mean of fluorescence intensity and Standard Error have been determined. Results MCL exhibited a strong expression of CD148 combined with a weak expression of CD180 and CD200. A weak expression of CD148 and CD180 coupled to a strong expression of CD200 was typical of the CLL group and a weak expression of CD148 and CD200 coupled to a strong expression of CD180 was observed in the MZL group. A moderate expression of these three markers was observed in the LPL group. A threshold corresponding to MFI +/- 4 standard error was then calculated for each group, and patients were categorized following the expression profile of these 3 markers (see figures). In this cohort, the above described profiles correctly identified MCL cases with a specificity of 92% and a sensitivity of 64%, aCLL cases with a specificity of 100% and a sensitivity of 47%, LPL cases with a specificity of 90% and a sensitivity of 54% and MZL cases with a specificity of 99% and a sensitivity of 60%. Conclusion These results strongly suggest that the incorporation of these three markers CD148 CD180 and CD200 in addition of the routinely used flow cytometry panel can be helpful in a number of cases for which the diagnosis is difficult. References: 1) Miguet et al leukemia 2013 2) Miguet et al journal of proteome research 2009 Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Interest of the CD148, CD180 and CD200 Combination in Flow Cytometry Analyses for Mature B-Cell Neoplasms Diagnosis

Blood ◽

10.1182/blood.v124.21.5407.5407 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5407-5407

Author(s):

Laurent Miguet ◽

Caroline Mayeur-Rousse ◽

Sarah Lennon ◽

Luc Fornecker ◽

Carine Gervais ◽

...

Keyword(s):

Flow Cytometry ◽

Differential Diagnosis ◽

B Cell ◽

Expression Profile ◽

Standard Error ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Strong Expression ◽

Group A ◽

Weak Expression

Abstract There are a number of small B-cell proliferations that do not fall into any of the types of B-cell neoplasms recognized in the WHO classification using classical immunophenotypic markers. This situation is notably encountered in the case of the differential diagnosis of marginal zone lymphoma (MZL), atypical chronic lymphocytic leukemia (aCLL) mantle cell lymphoma (MCL) or lymphoplasmacytic lymphoma (LPL). This is mostly due to the lack of immunological specific markers especially when histological samples are not available or during leukemic phases of atypical B-cell neoplasms. In order to find new markers to discriminate between these different malignancies, we have previously developed a proteomic strategy based on the analyses of plasma membrane microparticles and proposed two new specific markers: CD148 and CD1801, 2 for MCL and MZL respectively. The simultaneous use of these two markers, together with the CD200 that is positive in most cases of CLL and negative in MCL3 could be of great interest to better assess the differential diagnosis. In the present study, we report the results obtained by the combination of the three markers studied in addition to the routine flow cytometry panel: CD148 (Clone 143-41 FITC); CD180 (Clone G28.8 PE) and CD200 (Clone OX104 APC). An expression profile of these proteins have been established on a well characterized set of patients: CLL with a Matutes score > 3 (N=28); MCL harboring t(11;14) translocation or CCND1 overexpression (N=20); LPL (N=16) classified following cytological morphology, IgM peak and positivity of CD38, and MZL (N=27), displaying a CD5- CD23-immunophenotype associated to a splenomegaly. For each group the mean of fluorescence intensity and Standard Error have been determined. MCL patients exhibited a strong expression of CD148 (MFI = 1480) combined with a weak expression of CD180 and CD200 (MFI = 888 and 426 respectively). A weak expression of CD148 and CD180 (MFI = 495 and 754) coupled to a strong expression of CD200 (MFI = 3750) was typical of the CLL group and a weak expression of CD148 and CD200 (MFI = 640 and 1200) coupled to a strong expression of CD180 (MFI = 5300) was observed in the MZL group. A moderate expression of these three markers was observed in the LPL group. A threshold corresponding to MFI +/- 4 standard error was then calculated for each group (see table 1), and patients were categorized following the expression profile of these 3 markers. Table 1: threshold calculated from the average MFI and the associated standard error for each studied pathologies. CD148 CD180 CD200 MCL >980 <1500 <900 CLL <680 <1150 >500 MZL <1000 >2700 <2200 LPL 540 to 1350 500 to 1450 100 to 2700 In this cohort, the above described profiles correctly identified MCL cases with a specificity of 96% and a sensitivity of 65%, CLL cases with a specificity of 95% and a sensitivity of 79%, LPL cases with a specificity of 95% and a sensitivity of 31% and MZL cases with a specificity of 99% and a sensitivity of 52%. These results strongly suggest that the incorporation of these three markers CD148 CD180 and CD200 in addition of the routinely used flow cytometry panel can be helpful in a number of cases for which the diagnosis remains difficult. References: 1) Miguet et al. Leukemia 2013 2) Miguet et al. Journal of Proteome Research 2009 3) Palumbo et al. Leukemia Research 2009 Disclosures No relevant conflicts of interest to declare.

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CD180 Expression in B-Cell Lymphomas: A Multicenter Geil Study

Blood ◽

10.1182/blood.v124.21.1628.1628 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1628-1628

Author(s):

Caroline Mayeur-Rousse ◽

Julien Guy ◽

Laurent Miguet ◽

Sabrina Bouyer ◽

Franck Genevieve ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

University Hospital ◽

Routine Practice ◽

Similar Data ◽

B Cell Lymphomas ◽

Whitney Test ◽

Mann Whitney Test

Abstract CD180 is a Toll-Like Receptor homolog strongly expressed on normal human B-cells and involved in innate immune responses. Previous proteomic analyses on microparticles derived from mature B-cell neoplasms allowed us to identify CD180 as a marker of marginal zone lymphomas (MZL)(Miguet, Leukemia, 2013). Using flow cytometry on blood samples we showed that this protein is lost or underexpressed at the plasma membrane for almost all B-cell lymphomas except MZL. In order to confirm its clinical relevance, we conducted a prospective multicenter flow cytometry study in 5 French University Hospital laboratories, on behalf of the GEIL. Blood or bone marrow samples from 236 patients were studied (20 normal controls ; 74 chronic lymphocytic leukemia (CLL); 21 mantle cell lymphoma (MCL); 42 lymphoplasmacytic lymphoma (LPL); 13 follicular lymphoma (FL) ; 58 MZL, 14 of which with numerous villous lymphocytes; 8 hairy cell leukemia (HCL)). Analyses were performed either on FACSCanto II (BD Biosciences, 3 centers) or on Navios (Beckman Coulter, 2 centers) instruments. Harmonization process was performed using Rainbow beads (Spherotech). For the CLL group, CD180 Median fluorescence (MdFI) in each center was not significantly different (Anova test, p>0.05). Instruments’ harmonization was therefore effective enough to obtain similar data from all centres. In the whole cohort, CD180 was significantly less expressed in the group of lymphomas -including CLL, MCL, LPL and FL- than in controls (Mann-Whitney test, p<0.05). Conversely, in the group of MZL and HCL, CD180 MdFI was not different from those of controls (Mann-Whitney test, p>0.05) but significantly higher than in CLL, MCL, LPL and FL (Mann-Whitney test, p<0.0001). Distinction between MZL and lymphomas with numerous villous lymphocytes was possible (Mann-Whitney test, p=0.0012) but not between MZL and HCL. ROC curve analysis determined a CD180 MdFI threshold of 1800 which allow the positive diagnosis of MZL with a sensitivity of 77% and specificity of 92%. These results underline the efficiency of CD180 as a single positive and robust marker for MZL diagnosis, and confirm that between centers and between instruments harmonization is largely feasible in routine practice as published recently (Solly F et al. Cytomery part A, 2013). It should be emphasized that among the group of lymphomas with intense expression of CD180, all interestingly originating from the spleen, those with numerous villous lymphocytes display the highest expression. We described for the first time in this study the strong positivity of CD180 in HCL. Anti-CD180 antibody may be included in diagnosis combination markers in order to improve the diagnosis of chronic B-cell malignancies Disclosures No relevant conflicts of interest to declare.

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Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study

Blood Advances ◽

10.1182/bloodadvances.2021005725 ◽

2021 ◽

Author(s):

Sebastian Böttcher ◽

Robby Engelmann ◽

Georgiana Grigore ◽

Paula Carolina Fernandez ◽

Joana Caetano ◽

...

Keyword(s):

Flow Cytometry ◽

Differential Diagnosis ◽

Follicular Lymphoma ◽

B Cell ◽

Diagnostic Algorithm ◽

Lymphocytic Leukemia ◽

B Cell Lymphomas ◽

Multicentric Study ◽

Predictive Values ◽

Large B Cell

Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing nine disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis we subsequently utilized Canonical Correlation Analysis of 176 training cases to project the multi-dimensional space of all 26 immunophenotypic parameters into 36 two-dimensional plots for each possible pair-wise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%) and mantle cell lymphoma (95.4%). Burkitt and CD10+ diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD10- diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms.

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Multiparameter Flow Cytometry (MFC) for Identification of the Waldenström's Clone in IgM MGUS and Waldenstrom's Macroglobulinemia (WM): New Criteria for Differential Diagnosis and Risk Stratification

Blood ◽

10.1182/blood.v120.21.936.936 ◽

2012 ◽

Vol 120 (21) ◽

pp. 936-936

Author(s):

Bruno Paiva ◽

Maria-Carmen Montes ◽

Ramón García-Sanz ◽

Jennifer Alonso ◽

Natalia de las Heras ◽

...

Keyword(s):

Flow Cytometry ◽

Differential Diagnosis ◽

B Cells ◽

B Cell ◽

Light Chain ◽

Conflicts Of Interest ◽

Phenotypic Characterization ◽

International Prognostic Scoring System ◽

Multiparameter Flow Cytometry ◽

Risk Of Progression

Abstract Abstract 936 Demonstration of bone marrow (BM) infiltration by lymphoplasmacytic lymphoma is essential to the diagnosis of WM, and a trephine biopsy is considered mandatory for this assessment. Multiparameter flow cytometry (MFC) has demonstrated its clinical relevance in MGUS and myeloma; however, immunophenotypic studies on IgM monoclonal gammopathies are scanty, and focus only in patients with WM. Herein, MFC immunophenotyping was performed on BM samples from 244 patients, including 67 IgM MGUS, 77 smoldering, and 100 symptomatic WM newly diagnosed patients according to the Second International Workshop. A four color panel that systematically allowed the identification of B cells and plasma cells (PC), and their phenotypic characterization for a total of 24 antigens was used. We first analyzed the percentage of B cells and PC in BM and the percentage of light chain restricted cells in both compartments. Our results show a progressive increment of B cells from IgM MGUS to smoldering and symptomatic WM (medians of 2%, 9% and 12%; P<.001), as well of light chain restricted B cells (75%, 96% and 99%; P<.001). In contrast, no differences were found for the percentage of PC (median of 0.3%), but light chain restricted PC progressively increased from IgM MGUS to smoldering and symptomatic WM (70%, 85% and 97%; P<.001). Accordingly, only 1% of IgM MGUS patients showed >10% B cells, in contrast to 34% and 55% of smoldering and symptomatic WM (P<.001). Likewise, only 1% of IgM MGUS patients showed 100% light chain restricted B cells, in contrast to 19% and 40% of smoldering and symptomatic WM (P<.001); similar results being also found using a cutoff of 100% light chain restricted PC. Subsequently, we explored whether the percentages of BM and light chain restricted B cells and PC could predict time to progression (TTP) from smoldering into symptomatic WM, as well as overall survival (OS) in symptomatic WM. In smoldering WM, B cells (>10% vs ≤10%: median TTP of 47m vs 145m; P=.016) and light chain restricted B cells (100% vs <100%: 26m vs 145m; P<.001) but not PC, predicted risk of progression. On the multivariate analysis that included serum M-spike (±3g/dL), BM infiltration (±50% lymphoplasmacytic cells), BM B-cells and light chain restricted B cells (by MFC), only the later retained independent prognostic value (HR: 19.8, P=.001). Upon analyzing factors influencing survival in symptomatic WM patients, cases with >10% B cells showed a trend for inferior OS (P=.080), and significant differences emerged when comparing patients with 100% vs <100% light chain restricted B cells (median OS 44m vs 78m; P=.001). The later marker was independent (HR: 2.6; P=.004) of the International Prognostic Scoring System (HR: 2.2; P=.006). Focusing on the antigenic profiles of B cells and PC, we noted that within the B-cell compartment there was a progressive increment of CD22dim (69%, 92% and 88%; P<.001), CD25+ (61%, 88% and 90%; P<.001) and sIgM+ (88%, 95% and 97%; P=.002) B cells from IgM MGUS to smoldering and symptomatic WM. This underlies that the accumulating light chain restricted clonal B cells show a characteristic Waldenstrom's phenotype (CD22dim/CD25+/IgM+). Of note, a bimodal (from - to +) expression for the B cell memory marker CD27 was found in >50% of WM patients, which raises the possibility that the WM clone may arise, at least in some cases, before antigenic stimulation; subsequent maturation of the clone into PC would explain the typical presence of somatic hypermutations. On the other hand, B-cells from IgM MGUS and WM patients were negative in ≥90% of cases for CD5, CD10, CD11c and CD103, which can be useful to differentiate between WM and other B-NHL. Finally, the antigenic profile of PC in IgM MGUS and WM was similar to that of normal PC, and different from myeloma PC by consistently showing a CD27+ and CD56- phenotype, in addition to sIgM+ expression in ≥87% of all cases. Similarly to B-cells, we also noted that within the PC compartment there was a progressive increment of CD19+, CD45+ and sIgM+ CD20+ PC from IgM MGUS to smoldering and symptomatic WM. This underlies that this transition is asssociated with an accumulation of light chain restricted clonal PC displaying an immature/plasmablastic phenotype. In summary, our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM. Disclosures: No relevant conflicts of interest to declare.

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The Earliest Activation Marker CD69 Is An Independent and Strong Progression Indicator in B-Cell Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.2359.2359 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2359-2359

Author(s):

Giovanni Del Poeta ◽

Maria Ilaria Del Principe ◽

Cristina Simotti ◽

Francesco Buccisano ◽

Luca Maurillo ◽

...

Keyword(s):

Flow Cytometry ◽

Multivariate Analysis ◽

Prognostic Factor ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Significant Prognostic Factor ◽

Therapeutic Decisions ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abstract 2359 Poster Board II-336 B-cell chronic lymphocytic leukemia (B-CLL) exhibits features of activated and antigen-experienced B-lymphocytes and CD69 overexpression resembles B cells at an earlier and greater state of activation (Damle, 2002 and 2007). Moreover, the recent in vitro generation of anti-CD69 monoclonal antibodies able to inhibit either tumor growth or enhance NK cell function (Esplugues, 2005) prompt us to extensively evaluate CD69 antigen in our B-CLL cases. The primary endpoints of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD69; 2) to assess the additive prognostic value of CD69 and ZAP-70 and finally 3) to confirm CD69 as an independent prognostic factor. We investigated 401 patients (pts), median age 65 years, 219 males and 182 females. With regard to modified Rai stages, 120 pts had a low stage, 263 an intermediate stage and 18 a high stage. ZAP-70 and CD69 were determined by multicolor flow cytometry, fixing the cut-off values > 20% and >30%, respectively. ZAP-70+ and CD69+ pts were 166/401 (41%) and 108/401 (27%), respectively. CD69 lower than 30% was significantly associated with low Rai stage (105/120; P<0.0001), lymphocyte doubling time >12 months (258/332; P=0.00001), beta-2 microglobulin (B-2M) <2.2 mg/dl (181/223; P=0.00004) and soluble CD23 <70 U/ml (196/240; P<0.00001). There were significant correlations between lower CD69 and IgVH gene mutated status (283 total cases, 150/210; P=0.0001) or low risk (normal or 13q-) FISH cytogenetics (296 total cases, 149/213; P<0.00001). Equally, a strict association was found between lower CD69 and lower ZAP-70 (185/293; P=0.0003). With regard to clinical outcome, both a shorter PFS and OS were observed in ZAP-70+ pts (6% vs 56% at 12 years and 30% vs 95% at 16 years; P<0.00001) as well as in CD69+ pts (9% vs 49% at 14 years, P<0.00001 and 49% vs 75% at 16 years; P=0.00002). Noteworthy, ZAP-70 and CD69 showed additive prognostic properties, since ZAP-70 <20% plus CD69 <30% identified a B-CLL subset at better prognosis with regard to PFS (65% vs 2% at 12 years; P<0.00001, Figure) and OS (97% vs 35% at 14 years; P<0.00001). The two discordant subsets (CD69+ZAP-70 negative and CD69-ZAP-70+) showed an intermediate outcome (Figure). In multivariate analysis of PFS, CD69 (P=0.001) and ZAP-70 (P=0.005) together with cytogenetics and B-2M were confirmed to be independent prognostic factors. On the other hand, with regard to multivariate analysis of OS, age > or < 60 years resulted to be the most significant prognostic factor (P=0.004) followed by CD38 (P=0.01), IgVH status (P=0.02) and ZAP-70 (P=0.04). In conclusion, CD69 antigen, determined by flow cytometry, should be considered a novel important prognostic parameter in B-CLL and its easy and rapid laboratory determination may allow us to identify early progressive pts in order to take timely therapeutic decisions. Disclosures: No relevant conflicts of interest to declare.

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Identification of a Novel B-CLL Subset Expressing Mutated Stereotyped B-Cell Receptors with Specificity for Yeast Mannan

Blood ◽

10.1182/blood.v118.21.623.623 ◽

2011 ◽

Vol 118 (21) ◽

pp. 623-623

Author(s):

Robbert Hoogeboom ◽

Kok P. van Kessel ◽

Thera A.M. Wormhoudt ◽

Roy J.A. Reinten ◽

Ludo M.E. Evers ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Wall ◽

B Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Hair Follicles ◽

Lymphocytic Leukemia ◽

Light Chains ◽

Cervical Smears ◽

Leukemic Clone

Abstract Abstract 623 The IgVH/IgVL repertoire of the leukemic clones in B-cell chronic lymphocytic leukemia (B-CLL) patients is biased as compared to the repertoire of various B-cell subsets in healthy donors. Approximately, 30% of B-CLL patients can be classified in homology subsets based on the B-cell receptor (BCR) heavy chain complementary determining region 3 (CDR3) amino acid sequence of their leukemic clone. Interestingly, within these homology subsets, recurrent somatic hypermutations have been identified and heavy chains are paired to characteristic light chains. The occurrence of B-CLL groups expressing highly similar BCRs strongly suggests a shared specificity and involvement of recurrent antigens in the development of B-CLL. We have identified a novel B-CLL homology subset of four patients with a leukemic clone expressing highly similar mutated IgVH coupled to nearly identical IgVL rearrangements. Interestingly, all four patients had subset-biased somatic replacement mutations at two positions in IgVH. We produced the BCR of three patients belonging to this homology subset as recombinant soluble IgM (Subset-IgM) as well as BCRs of twenty-six cases expressing other rearrangements (Control-IgM). The Subset-IgM did not show any signs of autoreactivity in tissue arrays and were not found polyreactive by ELISAs for various well-known autoantigens. Therefore, we tested for reactivity with common pathogens by flow cytometry. The Subset IgM did not stain any of 20 bacterial strains. Interestingly, the yeast Candida Albicans stained brightly with Subset-IgM, as well as two other Candida strains and zymosan (cell wall of Saccharomyces Cerevisiae) (Figure 1A). None of the twenty-six Control-IgM showed this specificity. The Subset-IgM also stained yeast-infected hair-follicles and cervical smears by immunohistochemistry (Figure 1B). By ELISA, the antigen was found to be mannan, a main constituent of the yeast cell wall. Preincubation of Subset-IgM with mannan abolished the reactivity to Zymosan. Furthermore, B-CLL cells of subset patients bound zymosan ex-vivo. Of note, when the subset characteristic light chain was replaced by light chains of control B-CLL, reactivity to mannan was abrogated. In addition, when cultured in vitro, mannan induced proliferation of B-CLL cells of this homology subset, whereas proliferation of control B-CLL cells was not induced. In conclusion, we identified a novel B-CLL homology subset, expressing highly similar BCRs with specificity for yeast mannan. This study shows for the first time that a hypermutated B-CLL homology subset expresses functional BCRs with high-affinity towards a pathogen, which are able to transduce signals that support tumour cell growth. Figure 1: Recombinant IgM of a novel B-CLL subset stain yeast (A) Subset-IgM stain Zymosan by flow cytometry. (B) Staining of yeast in infected cervical smears. Left: Control-IgM. Right: Subset-IgM. Arrows indicate yeast (in hyphal form). Figure 1:. Recombinant IgM of a novel B-CLL subset stain yeast . / (A) Subset-IgM stain Zymosan by flow cytometry. (B) Staining of yeast in infected cervical smears. Left: Control-IgM. Right: Subset-IgM. Arrows indicate yeast (in hyphal form). Disclosures: No relevant conflicts of interest to declare.

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CD200 Expression May Help in Differential Diagnosis between Mantle Cell Lymphoma (MCL) and B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽

10.1182/blood.v110.11.4672.4672 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4672-4672

Author(s):

Giuseppe A. Palumbo ◽

Giovanna Forgione ◽

Nunzia Parrinello ◽

Katia Cardillo ◽

Annalisa Chiarenza ◽

...

Keyword(s):

Flow Cytometry ◽

Differential Diagnosis ◽

B Cell ◽

Cyclin D1 ◽

Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Neoplastic Cells ◽

Exact Test ◽

Mantle Cell ◽

Cell Chronic Lymphocytic Leukemia

Abstract Mantle cell lymphoma (MCL) is a B-cell malignancy that shares many morphologic and immunophenotyping features with B-cell chronic lymphocytic leukemia (B-CLL). However, these two entities are characterized by a very different prognosis and response to therapies. Differential diagnosis is assessed by a panel of monoclonal antibodies (moAbs) to screen the expression of CD5, CD23 and Cyclin D1. Nevertheless, some of these antibodies work better on fresh or frozen tissues; for example, on fixed tissues (expecially by B5) CD5 and Cyclin D1 can give equivocal or even negative results. In addition, it is cumbersome to evaluate Cyclin D1 by flow cytometry. CD200 (previously referred to as OX2) is a membrane glycoprotein, belonging to the immunoglobulin superfamily, expressed on T and B lymphocytes (but not on NK cells) and it might play an immunosuppressive role. Its expression has also been reported on human myeloma plasmacells and B-CLL cells. We investigated the expression of CD200 on fresh neoplastic cells of 90 patients (82 B-CLL and 8 MCL in leukemic phase) by flow cytometry, using a mouse IgG1 anti-human moAb (MRC OX-104, BD Pharmingen, U.S.A.). In our series, CD200 was present on neoplastic cells of all 82 B-CLL (expression on 62–100% of CD5+ cells, mean 96%, standard deviation 7%). On the contrary, in MCL patients (Cyclin D1+ and/or t(11;14) FISH+) CD200 was positive in less than 20% of CD5+ cells in 3 subjects (4–18%) and totally absent in the remaining 5 (two-sided Fisher’s exact test p<0.0001). To further extend the study, we examined CD200 by immunohistochemistry on paraffin-embedded formalin-fixed lympoid tissues and Lowy-fixed bone marrow (BM) trephine biopsies from 18 B-CLL (12 lymphnodes and 7 BM samples) and 19 MCL (11 lymphoid tissues and 9 BM) patients, using a goat anti-human IgG affinity-purified polyclonal antibody (R&D Systems, U.S.A.). Again, all B-CLL neoplastic cells were positive for CD200 both in lymphnodes and in BM while all MCL cells were negative, both in lympoid tissues and in BM (two-sided Fisher’s exact test p<0.0001). Notably, in all MCL CD200-negative lympoid tissue biopsies, it was possible to observe CD200+ residual dendritic cells (useful as immunohistochemistry reaction positive control). Moreover, results were consistent in old archival samples and even in non-perfectly prepared tissues referred to our Center. We would propose to add CD200 in flow cytometry and immunohistochemistry routine panels, as it can be of diagnostic utility in distinguishing between MCL and B-CLL, in particular in mantle cell leukemia patients. Finally, these data have to be considered in view of the proposed targeted therapy with anti-CD200, to eventually exclude MCL patients.

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The role of immunophenotyping in differential diagnosis of chronic lymphocytic leukemia

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh1404197d ◽

2014 ◽

Vol 142 (3-4) ◽

pp. 197-203 ◽

Cited By ~ 3

Author(s):

Tijana Dragovic-Ivancevic ◽

Nada Kraguljac-Kurtovic ◽

Vesna Knezevic ◽

Andrija Bogdanovic ◽

Biljana Mihaljevic ◽

...

Keyword(s):

Flow Cytometry ◽

Differential Diagnosis ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

De Novo ◽

Expression Patterns ◽

Lymphocytic Leukemia ◽

Control Group ◽

Very High Frequency ◽

Marker Combination

Introduction. Accurate diagnosis of chronic lymphocytic leukemia (CLL) acquires immunophenotyping by flow cytometry in order to facilitate differential diagnosis between CLL and other mature B-cell neoplasms (MBCN). Objective. The aim of this study was to define immunological profile of CLL cells. Methods. Immunophenotyping by flow cytometry was performed on peripheral blood specimens at diagnosis in the group of 211 patients with de novo MBCN. Results. Absolute count of B-cells was significantly increased in all MBCN patients comparing to healthy control group (p<0.05). B-cell monoclonality was detected in 96% of all MBCN patients, by using surface immunoglobulin (sIg) light chain restriction. B-cell antigens, CD19, CD20, CD22, were expressed with very high frequency in CLL and other MBCN. In comparison with other MBCN, in CLL group, the frequency of expression was higher for CD5 and CD23 (p<0.0001), though lower for FMC7 antigen (p<0.0001). CLL patients were characterized by lower expression patterns of CD20, CD22, CD79b, and sIg (p<0.0001) as well as higher expression pattern of CD5 antigen (p<0.05). Correlation between the final diagnosis of MBCN and values of CLL scoring system showed that the majority of CLL patients (97%) had higher values (5 or 4) whereas the majority of other MBCN patients (96%) had lower score values (0-3). Conclusion. Our results have shown that characteristic immunophenotype which differentiates CLL from other MBCN is defined by following marker combination - CD19+ CD20+low CD22+low CD5+high CD23+ FMC7- CD79b+low sIg+low. CLL score values of 5 or 4 points are highly suggestive for diagnosis of CLL.

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The Role of Molacular Genetic Examination in Differential Diagnosis and Prognostic Prediction in Patints with B-Cell Chronic Lymphoproliferative Disorders: A Restrospective Study of 1592 Patients

Blood ◽

10.1182/blood-2018-99-114920 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5329-5329

Author(s):

Yuting Yan ◽

Wenjie Xiong ◽

Shuhua Yi ◽

Li Zengjun ◽

Dehui Zou ◽

...

Keyword(s):

Differential Diagnosis ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoproliferative Disorders ◽

Lymphocytic Leukemia ◽

Initial Time ◽

Female Ratio ◽

Male Predominance ◽

Biopsy Specimens ◽

Prognostic Prediction

Abstract Background: B-cell chronic lymphoproliferative disorders (BCLPD) are a heterogeneous group of mature B-cell malignancies characterized by monoclonal proliferation in the bone marrow (BM) or peripheral blood (PB). The overlapped clinical features and the lack of individual diagnostic markers make diagnosis challenging, especially in patients who have difficulty to get adequate non-BM biopsy specimens or patients manifest as leukemic onset without nodes-involvement. It is necessary to explore novel methods to make precise diagnostic classification and prognostic prediction through simply PB/BM examination. Methods and results: A total of 1592 patients with newly diagnosed BCLPD were enrolled from January 1998 to December 2016, of which chronic lymphocytic leukemia (CLL) accounted for 39%, leukemic marginal zone lymphoma (l-MZL)occupied 13%, and Waldenstrom's macroglobulinemia/ lymphoplasmacytic lymphoma (LPL/WM), leukemic follicular lymphoma (l-FL) and mantle cell lymphoma (l-MCL) were 13%, 9% and 8%, respectively. There are 14% patients failed to fit into any of the categories and remaining unclassified which defined as BCLPDU. The median age at diagnosis was 58 years old. The male/female ratio of all BCLPD patients was 1.84:1. Male predominance was pronounced in WM/LPL, l-MCL and HCL with a M/F ratio of 2.5-2.7, while with balance gender in l-MZL and l-FL. FISH analyses were performed in 1234 patients. 17p and 11q deletion were most common in l-MCL (36% and 17%), while 13q deletion and trisomy 12 were most frequent in CLL (35% and 21%). Patients with 17p deletion showed significantly adverse survival in CLL and l-MCL (p<0.05), but the phenomenon was not found in other types of BCLPD. Instead, complex karyotype showed better predictive capability of worse survival than 17p deletion in patients with l-FL, l-MZL and BCLPDU. A total of 665 patients had been successfully amplified the IGHV-IGHD-IGHJ rearrangements. Based on a cut-off value of 98% homology, the IGHV mutated proportion in patients with l-MCL was 48.6%, which was higher than other BCLPDs, following CLL 33.3% and l-MZL 28.6%, while most of WM and l-FL were unmutated. The most frequent rearrangement in l-MZL were IGHV1-2*01, which was seldom found in other subtypes. Also, DH2-2 and VH3-7 were mainly found in CLL and l-MCL respectively. Besides, we have identified several stereotyped IGH CDR3 which were specific for the certain BCLPD subtype. About 23% unclassified patients could acquire further diagnosis according to the special mutation status, VDJ rearrangements or stereotyped CDR3 sequencing. With a median follow-up of 50.3 months, 522 patients (38.0%) had disease progression or died. Patients with lMCL have a significant worse survival than other entities, with a 3-year overall survival (OS) rate of 58%. Following , patients with WM/LPL had relatively poorer prognosis with a 5-year OS rate of 68.2%, while the prognosis for l-MZL, l-FL and CLL were relatively favourable, with a 5-year OS of 80%.The OS of the B-CLPD had improved over time due to the emergence of immunotherapy and novel (3-year OS improved from 82.1% to 92.2%). The improvement of l-MCL and WM was obvious but not for CLL and l-MZL. Conclusion: Non-BM biopsy specimens are not available for part of BCLPD patients. Overlapped magnification of BM/PB brings on difficulties in differential diagnosis. Besides morphology and immunophenotype, comprehensive analysis of FISH, karyotype, IGHV, MYD88 or BRAF mutation and other molecular examination might contribute to further differential diagnosis and prognostic prediction. Moreover, it might provide advice for the initial time and strategy of treatment. Disclosures No relevant conflicts of interest to declare.

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Anlotinib Shows Potent Antileukemia Effects in B-Cell Acute Lymphocytic Leukemia through the Blockage of Angiogenic Related Pathways

Blood ◽

10.1182/blood-2020-141039 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 49-49

Author(s):

Qiuling Chen ◽

Yuelong Jiang ◽

Qinwei Chen ◽

Long Liu ◽

Bing Xu

Keyword(s):

B Cell ◽

Solid Tumors ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Unmet Need ◽

Lymphocytic Leukemia ◽

Advanced Lung Cancer ◽

Antitumor Effects

Acute lymphoblastic leukemia (ALL) derives from the malignant transformation of lymphoid progenitor cells with ~85% being originated from B-cell progenitors (B-ALL). Despite fairly good prognoses for most pediatric B-ALL patients, the outcome is fatal in over 50% of adult patients who have a recurrent or progressive disease and lack of effective therapeutic approaches. Therefore, novel treatment strategies with high efficacy and low toxicity are an unmet need for B-ALL patients, especially those with relapsed or refractory status. Angiogenesis is a process of new vessel formation that requires the participation of multiple proangiogenic factors (e.g., VEGF, PDGF, and FGF) and their corresponding receptors (e.g., VEGFR, PDGFR, and FGFR). Angiogenesis, a well-established feature of solid tumors, also contributes to leukemia progression and correlates with the involvement of specific sanctuary sites in ALL, highlighting that the perturbation of angiogenesis would be an attractive approach for ALL treatment. Anlotinib is an oral tyrosine kinase (TKI) inhibitor with a broad range of antitumor effects via the suppression of VEGFR, PDGFR and FGFR. Of importance, anlotinib has been approved for the treatment of advanced lung cancer in China. Here, we evaluated the antileukemia activity of anlotinib in preclinical B-ALL models and its underlying molecular mechanisms. In this study, we observed that anlotinib significantly blunted the capability of cell proliferation and arrested cell cycle at G2 phase in B-ALL cell lines. Subsequently, we found that anlotinib resulted in remarkably enhanced apoptosis in B-ALL in vitro. To assess the in vivo antileukemia potential, we established a B-ALL patient-derived xenograft (PDX) mouse model and then treated the B-ALL PDX model with anlotinib. As a result, oral administration of anlotinib pronouncedly delayed in vivo B-ALL cell growth and reduced leukemia burden with acceptable safety profiles in this model. As for the mechanism of action, the antileukemia effect of anlotinib was associated with the disruption of the role of VEGFR2, PDGFRb, and FGFR3. Moreover, we revealed that this drug blocked the PI3K/AKT/mTOR/ signaling, a pathway that is linked with angiogenesis and its proangiogenic regulators, including VEGFR2, PDGFRb, and FGFR3. In aggregate, these results indicate that anlotinib is a potent antitumor agent for the treatment of B-ALL via the inhibition of angiogenic relevant pathways, which provide a novel potential treatment intervention for patients with B-ALL who have little effective therapy options. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Anlotinib originally designed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

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