Correlation of Peripheral Blood Involvement Measured by Four-Color Flow Cytometry and the Achievement of Molecular Response in Mantle Cell Lymphoma Treated within the Prospectively Randomized Intergroup Trials of the European MCL Network

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1284-1284
Author(s):  
Christiane Pott ◽  
Sebastian Boettcher ◽  
Stefan Gesk ◽  
Reiner Siebert ◽  
Wolfram Klapper ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mantle Cell Lymphoma ◽  
Peripheral Blood ◽  
Cell Lymphoma ◽  
Bone Marrow Infiltration ◽  
Molecular Response ◽  
Clinical Parameters ◽  
Extranodal Involvement ◽  
Mantle Cell

Abstract Two prospectively randomized intergroup trials of the European MCL Network investigating the impact of different combined immuno-chemotherapy protocols for patients with stage II–IV mantle cell lymphoma (MCL) >< 65 yrs followed by autologous stem cell transplantation (PBSCT) for patients <65 yrs and a rituximab or interferone maintenance treatment for patients >65 yrs are currently performed. Patients and methods: 180 German patients with diagnostic peripheral blood (PB) involvement detectable by consensus IGH PCR were analysed for t(11;14) translocation by FISH and 4-colour Flow Cytometry (FC) at diagnosis as well as molecular response (MR) by quantitative IGH-RQ-PCR after induction. The results were compared to clinical parameters at diagnosis and PFS. Results: Patients had a median age of 61 years, an elevated LDH in 37%, B-symptoms in 41% and extranodal involvement in 37%. According to the MIPI risk factor score 26% had an adverse, 34% an intermediate and 40% a good prognosis. PB involvement by MCL was demonstrated in 67% (74/111) by t(11;14) FISH and in 97% (152/156) by FC. Detection of MCL cells by FC in PB did not correlate with clinical parameters as stage, LDH, extranodal involvement or bone marrow infiltration. However, median % of MCL cells measured by FC differed significantly with clinical stage (p<0.0001), LDH (p=0.0096) bone marrow infiltration (<0.0001) and MIPI prognostic index (0.0002). MR after induction treatment was achieved in 60% (60/100) of patients and correlated with BM infiltration at diagnosis (p=0.0117). There was a trend for improved PFS in patients achieving MR (PFS 90% vs. 75% at 15 months after induction) however, the observation time is still too short for definitive evaluation. Conclusion: FC is highly sensitive for detection of PB involvement in MCL and improves staging accuracy. Achievement of MR might be an early indicator for treatment outcome after immuno-chemotherapy in MCL with the potential of defining prognostic subgroups.

scholarly journals Minimal Residual Disease Monitoring By 8-Color Flow Cytometry in Mantle Cell Lymphoma Is Complementary to Q-PCR Monitoring and Will Facilitate Pre-Emptive Treatment: An EU-MCL and Lysa Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1657-1657
Author(s):  
Morgane Cheminant ◽  
Stephanie Schmit ◽  
Aurore Touzart ◽  
Coralie Derrieux ◽  
Marie-Hélène Delfau-Larue ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mantle Cell Lymphoma ◽  
Patient Management ◽  
Residual Disease ◽  
Cell Lymphoma ◽  
Clinical Relapse ◽  
Color Flow ◽  
Mantle Cell ◽  
Minimal Residual

Abstract Introduction: Mantle Cell Lymphoma (MCL) is characterized by frequent blood and bone marrow involvement. It has been demonstrated that use of Minimal Residual Disease (MRD) quantification in blood and/or bone marrow might be helpful in patient management. Gold standard MRD is based on Q-PCR clone specific amplification of IgH VDJ or IgH-BCL1 rearrangements, but these are relatively complex and time consuming and over half of the positive results are in a grey zone of borderline positivity. Flow cytometry (FCM) is more rapid and better adapted to individual patient management if quantitatively reproducible, but insufficiently sensitive when only 4 colors are used. We therefore developed a universal, 8-color, EuroFlow inspired, FCM strategy, which we compared with classical Q-PCR MRD in 61/97 patients included in (and 1 treated according to) the EU-MCL Younger and Elderly prospective trials who underwent Q-PCR MRD monitoring at Necker Hospital. Method: Q-PCR MRD from IgH VDJ (n=92) or BCL1-IgH (n=5) was performed prospectively from ficolled blood (PB) or bone marrow, from which residual material was cryopreserved in DMSO for FCM quantitation, using 10 antibodies labelled with 8 fluorochromes for positive and negative (CD45, CD19, CD5, LAIR1, CD11a, IGK, IGL, CD3, CD14 and CD56) gating, after diagnostic phenotyping of fresh material, using the same panel and a EuroFlow B lymphoid screening tube. Sensitivity of both techniques was at least 0.01% (1E-04). FCM was only considered positive if above 0.01%, whereas Q-PCR results were considered positive below quantifiable range (BQR) if borderline, above sensitivity, within Euro-MRD criteria for MRD positivity. BQR samples were separated based on the number of positive, triplicate samples. The objectives were to compare the two techniques and to determine their suitability for regular screening, with a view to pre-emptive treatment on molecular or phenotypic (MRD) relapse. Two patients were treated with Rituximab at MRD relapse, prior to clinical relapse, as proof of principle. Results: A total of 302 blood or bone marrow samples from 62 patients were quantified. Overall, 79% (42/53) of samples positive at or above 0.01% by PCR were also positive by FCM, compared to 29% (19/65) of those below 0.01%, but with at least 2 positive triplicates and virtually none of those with only 1 or no results above sensitivity (1%, 2/184). Quantification of the paired MRD results positive with PCR and/or FCM were significantly correlated (r2=0.74, P<0.0001). Amongst the 62 patients, 30 have relapsed and 19 have died. Nine relapsing patients (including one off protocol patient treated and monitored at initial and second MRD relapses) had sufficient MRD points to assess the capacity of PB Q-PCR or FCM to predict future clinical relapse sufficiently to justify pre-emptive treatment at MRD relapse. Clinical relapse was preceded by MRD relapse in 9/10 relapses by Q-PCR and 7/9 by FCM. Six of the 9 relapsing patients had achieved Q-PCR negativity in at least one PB sample. The mean latency for prediction by Q-PCR, when considering any increase in positivity to at least 2 positive triplicates as positive, was 11.3 months (range 1-24mths) and 5.4 months (0.5-11) when only results above 0.01% were considered positive. The equivalent latency by FCM was slightly shorter, at 6.5 months (0.5-21) Pre-emptive treatment of 2 patients at MRD relapse, prior to clinical relapse allowed re-establishment of molecular complete remission and a durable second remission in at least one with sufficient follow-up (Cf Fig.). Figure 1 Figure 1. Conclusion: Eight color flow cytometry is a promising alternative to classical clone-specific Q-PCR strategies in monitoring therapy in MCL, with an excellent correlation (29/31, 94%) for MRD levels of at least 0.1% and acceptable correlation at 0.01-0.1% (13/22, 59%). While less sensitive at very low levels on cryopreserved material, FCM may clarify the clinical relevance of low-level borderline positivity; however it remains to be determined prospectively which technique will have greater prognostic value in patient management. FCM sensitivity will be improved by prognostic testing of fresh whole blood or bone marrow, and this pilot data clearly justifies such studies. Finally, MRD relapse precedes clinical relapse by several months, justifying pre-emptive treatment, monitored by prospective FCM and IgH Q-PCR within clinical trials. Disclosures Dreyling: Roche: Honoraria, Research Funding.

Burkitt Transformation of Mantle Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4129-4129
Author(s):  
Kavita S. Reddy ◽  
Mohammad Ansari-Lari ◽  
Bruce Dipasquale
Keyword(s):  
Bone Marrow ◽  
Mantle Cell Lymphoma ◽  
Peripheral Blood ◽  
Blood Smear ◽  
Cell Lymphoma ◽  
Peripheral Blood Smear ◽  
Neoplastic Cells ◽  
Ki 67 ◽  
Mantle Cell ◽  
Neoplastic Lymphocytes

Abstract MYC rearrangements are not included as a genetic change in the blastoid variants of mantle cell lymphoma (Jaffe, et al (2001) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press.). We present two cases both with CCND1/IGH and MYC rearrangements. Case 1. An 82-year-old male with no known history of lymphoma presented with thrombocytopenia, loss of appetite and “abdominal fullness.” Imaging studies showed enlarged retroperitoneal lymph nodes. The peripheral blood smear had 18,000 WBC with approximately 30% circulating atypical lymphocytes. Flow cytometric studies of the bone marrow revealed a surface kappa light chain restricted CD10+ B-cell population. A bone marrow biopsy showed >90% of marrow cellularity comprised of neoplastic lymphocytes. The neoplastic lymphocytes were small to intermediate in size with minimal amounts of dark blue cytoplasm and several cytoplasmic vacuoles (Burkitt like morphology). By immunohistochemical stains, the neoplastic cells were positive for CD20, CD43, CD10, BCL-6, and cyclin D1, weakly and focally positive for BCL-2, and negative for CD23. The Ki-67 proliferation fraction was ∼100%. An immunohistochemical stain for CD5 was predominantly negative with a possible very faint blush on a subset of neoplastic B-cells. The FISH tests on bone marrow interphases were positive for a CCND1/IGH, a variant MYC/IGH, a variant MYC-BA rearrangements and negative for BCL6-BA and BCL2/IGH rearrangements. The variant MYC/IGH pattern was 3xMYC, 3xIGH, 1xFusion signals and MYC-BA pattern was 2x5′MYCcon3′MYC, 1x3′MYC. rearrangements. The karyotype was 44∼45,XY,del(2)(q11.2q21),der(3;17)(p10;q10), der(5)t(3;5)(q12;q15), t(11;14) (q13;q32) [cp6]/46,XY[14]. Since the karyotype had a t(11;14) and two normal 8 chromosomes, a metaphase FISH was analyzed to localize the signals for the MYC/IGH probe. The MYC signal were on both normal 8 chromosomes, a fusion signal was on a F-G sized chromosome. While the IGH signals were on the normal 14, der(14) and der(11). This was consistent with a cryptic MYC/IGH fusion in a three way rearrangement between chromosomes 8, 11 and 14. Case 2. A 69-year-old male having had a kidney transplant in 2001 was on immunosuppressive therapy. He presented with severe leukocytosis, anemia and thrombocytopenia and weight loss of about 12 pounds over several months. A peripheral blood smear showed 74,000 WBC with approximately 30% blasts. Bone marrow biopsies revealed normocellular bone marrow (50% cellularity). Interspersed large neoplastic lymphoid cells were shown by immunohistochemical stains to be positive for CD20, BCL-1, weak positive for BCL-2 and a Ki-67 staining > 90%. Flow cytometry indicated that the neoplastic cells were positive for kappa and CD5 but negative for CD11c and CD23. Interphases FISH on peripheral blood was positive for a CCND1/IGH rearrangement. The karyotype was 42∼44,X,-Y,add(1)(p13), t(2;8)(p12;q24), der(2)t(2;15)(p25;q11.2),+3,del(9)(p22p24),+del(9)(p22p24), − 10, del(11)(q21q23), t(11;14)(q13;q32) , − 13, − 15, − 17,add(17)(p11.2)[cp7]/46,XY[17]. FISH confirmed a MYC rearrangement. Therefore, this case had both CCND1/IGH and MYC/IGK rearrangement. Concomitant occurrence of a CCND1/IGH and a MYC rearrangement is rare in lymphomas. In Mitelman database of chromosome aberrations in cancer 2007, Four cases had both a t(11;14) and a t(8;14) translocation and two cases had both a t(11;14) and a t(2;8) translocation. This study expands the repertoire of abnormalities seen in blastoid transformation of mantle cell lymphoma. Being cognizant of a possible MYC involvement in the transformation of mantle cell lymphoma and its exploration would influence therapy.

Diagnostic Value of CD200 Expression for the Differentiation Between Chronic Lymphocytic Leukemia (CLL), CLL with Increased Prolymphocytes (CLL/PL) and Mantle Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2660-2660
Author(s):  
Wolfgang Kern ◽  
Richard Schabath ◽  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Infiltration ◽  
Malignant Cells ◽  
Mantle Cell ◽  
Equity Ownership

Abstract Abstract 2660 Background: Chronic lymphocytic leukemia (CLL) is diagnosed by the immunophenotye as published by Matutes et al. with strong expression of CD5 and CD23, weak expression of sCD22, CD79b and immunoglobulins and negativity for FMC7. CLL is readily differentiated from mantle cell lymphoma (MCL) which shares CD5 positivity but features strong expression of sCD22, CD79b and FMC7. CLL with increased prolymphocytes (CLL/PL), which cytomorphologically features 5–55% prolymphocytes, displays an immunophenotype in between CLL and MCL. The cytogenetic finding of a t(11;14) separates MCL from CLL/PL. The analysis of CD200 expression has been suggested to improve differentiation between CLL and MCL based on a limited number of cases and has not yet been assessed in CLL/PL. Aims: To assess the diagnostic usefulness of CD200 expression in the flow cytometric assessment of CLL, CLL/PL and MCL. Methods: We studied 100 patients with CLL (n=59), CLL/PL (n=27) and MCL (n=14) for expression of CD200. t(11;14) was confirmed in all cases with MCL and excluded in all cases with CLL/PL. 37 were female, 63 male, median age was 71.2 yrs (range 39.3–87.1), sample material was peripheral blood (n=68) or bone marrow (n=32). CD200 was analyzed using the antibody clone MRC OX-104 conjugated to phycoerythrin (BD Biosciences, Franklin Lakes, NJ). Cells were rated positive if they expressed CD200 stronger than the cut-off set by an isotype control. Cases were rated positive if ≥20% of the pathologic population as identified based on coexpression of CD19 and CD5 expressed CD200. Results: Mean±SD % values for CD200 positive cells were 94.9±14.0% in CLL, 78.7±27.0% in CLL/PL and 6.6±13.3% in MCL. Thus, besides significant differences between MCL with low CD200 expression and the two other entities with high CD200 expression (p<0.001 for both), there was also a significant difference between CLL and CLL/PL (p=0.006) with higher values for CLL. The same was true for the analysis of mean fluorescence intensities (MFIs): CLL cases expressed CD200 stronger (mean±SD 5.8±2.9) as compared to CLL/PL cases (3.9±2.8, p=0.007) while MCL cases had clearly the lowest MFI (0.3±0.3, p<0.001 for comparisons to both CLL and CLL/PL). In order to use a more reliable parameter than MFI of the malignant cells per se, we included normal T-cells as controls and calculated the MFI ratio “malignant cells:normal T-cells”. This again resulted in the highest values for patients with CLL (mean±SD 14.8±9.0) although the difference to patients with CLL/PL (12.3±10.4) was not significant. Patients with MCL also for this parameter had clearly lower values amounting to 1.2±0.9 (p<0.001 for comparisons to both CLL and CLL/PL). We than tested whether a cut-off for the CD200 expression parameters would be discriminative of the three entities. Regarding positivity for CD200 expression based on a cut-off of 20% malignant cells we found the following rates of positivity for CD200: 58/59 (98.3%) in patients with CLL, 26/27 (96.3%) in patients with CLL/PL and 2/14 (14.3%) in patients with MCL. Thus, although there were overall significant differences in the CD200 expression the application of a 20% cut-off was not capable of completely differentiating CLL and CLL/PL on the one hand from MCL on the other hand. Importantly, the “misclassified” cases mostly were not near to the cut-off (11.5% in CLL, 2.8% in CLL/PL, 47.0% and 22.5% in MCL). To improve classification we applied a cut-off of 0.48 for the MFI of CD200 expression in malignant cells. All 59 cases with CLL and all cases with CLL/PL had MFIs >0.48, however, only 12/14 cases with MCL had MFIs <0.48. Thus, although this analysis reveals highly significant differences between the three entities (p<0.001) there remain two misclassified MCL cases. While in one of these 2 cases the encountered MFI of 0.84 was only slightly above the cut-off and low-level bone marrow infiltration of 1% may have contributed to imprecision of MFI determination this cannot be considered for the second case (MFI 1.26, bone marrow infiltration 8%). The use of a cut-off for the MFI ratio “malignant cells:normal T-cells” yielded no improvement in classification. Conclusions: Assessment of CD200 expression may be applied in the differential diagnosis of CLL, CLL/PL and MCL to predict MCL with high probability based on a low-level expression of CD200, however, the sensitivity of this approach is limited by the infrequent MCL cases with higher expression of CD200. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Prognostic Significance of Bone Marrow Trephine and Peripheral Blood Smears in 55 Patients with Mantle Cell Lymphoma

1996 ◽  
Vol 21 (1-2) ◽  
pp. 115-125 ◽  
Author(s):  
Stefania Pittaluga ◽  
Gregor Verhoef ◽  
Arnold Criel ◽  
Alex Maes ◽  
Johan Nuyts ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mantle Cell Lymphoma ◽  
Peripheral Blood ◽  
Cell Lymphoma ◽  
Prognostic Significance ◽  
Bone Marrow Trephine ◽  
Blood Smears ◽  
Mantle Cell ◽  
Peripheral Blood Smears

Prominent intrasinusoidal infiltration of the bone marrow by mantle cell lymphoma

2003 ◽  
Vol 34 (8) ◽  
pp. 789-791 ◽  
Author(s):  
Andre A Schenka ◽  
Randy D Gascoyne ◽  
Eliane Duchayne ◽  
Georges Delsol ◽  
Pierre Brousset
Keyword(s):  
Bone Marrow ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Mantle Cell

scholarly journals PRMT5 Inhibition Drives Therapeutic Vulnerability to BCL-2 Inhibition with Venetoclax and Provides Rationale for Combination Therapy in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 302-302 ◽  
Author(s):  
Fiona Brown ◽  
Yang Zhang ◽  
Claire Hinterschied ◽  
Alexander Prouty ◽  
Shelby Sloan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Combination Treatment ◽  
Cell Lymphoma ◽  
Targeted Agents ◽  
Mantle Cell ◽  
Anti Tumor Activity

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, defined by the t(11;14) translocation and comprises 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 5 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life. Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting the cell cycle, PRC2 activity, and signaling via the BCR and PI3K/AKT pathways. We have developed first-in-class selective inhibitors of PRMT5 and, in collaboration with Prelude Therapeutics, we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models of human MCL. Selective inhibition of PRMT5 in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to chromatin. We identified 136 newly emerged FOXO1-bound genomic loci following 48 hours of PRMT5 inhibition in the CCMCL1 MCL line by performing chromatin immunoprecipitation-seq analysis. These genes were markedly upregulated in CCMCL1 cells treated with the PRMT5 inhibitor PRT382 as determined by RNA-seq analysis. Among those genes, we identified and confirmed FOXO1 recruitment to the promoter of BAX, a pro-apoptotic member of the BCL2 family of proteins. Treatment of MCL cell lines (Granta-519, CCMCL1, Z-138, and SEFA) with the selective PRMT5 inhibitor PRT382 (10, 100nM) led to upregulation of BAX protein levels and induction of programmed cell death as measured by annexin V/PI staining and flow cytometry. We hypothesized that induction of BAX would trigger a therapeutic vulnerability to the BCL2 inhibitor venetoclax, and that combination PRMT5/BCL2 inhibitor therapy would drive synergistic cell death in MCL. Single agent and combination treatment with venetoclax and PRT382 was performed in eight MCL lines including a new cell line generated from our ibrutinib-refractory PDX model (SEFA) and IC50 and synergy scores were calculated. The Z-138 line was most sensitive to venetoclax (IC50&lt;10nM) while CCMCL-1, SP53, JeKo-1, and Granta-519 demonstrated relative resistance (IC50&gt;1uM). All lines reached an IC50 &lt;1uM when co-treated with PRT382, with IC50 values ranging from 20 - 500nM. Combination treatments showed high levels of synergy (scores &gt; 20) in 4 lines and moderate synergy (scores 10-20) in 2 lines. The two lines with the highest levels of synergy, Z-138 and SEFA, express high levels of BCL-2 and are Ibrutinib resistant. Overall there was a strong positive correlation between BCL2 expression and synergy score (r=0.707), and no correlation between PRMT5 expression and synergy score (r=0.084). In vivo evaluation in two preclinical MCL models (Granta-519 NSG mouse flank and an ibrutinib-resistant MCL PDX) showed therapeutic synergy with combination venetoclax/PRT382 treatment. In both models, mice were treated with sub-therapeutic doses of venetoclax and/or PRT543 (Granta) or PRT382 (IR-MCL PDX) and tumor burden assessed weekly via flank mass measurement (Granta) or flow cytometry (IR-MCL-PDX). Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in both MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. PRT543, a selective PRMT5 inhibitor, has been advanced into clinical studies for the treatment of patients with solid tumors and hematologic malignancies, including MCL (NCT03886831). Disclosures Zhang: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

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