Showing papers by "Constance M. Yuan published in 2016"

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study

Abstract: In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Disruption of in vivo Chronic Lymphocytic Leukemia Tumor-Microenvironment Interactions by Ibrutinib--Findings from an Investigator-Initiated Phase II Study

Abstract: Purpose: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment. Experimental Design: Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed. Results: Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4 + T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4 + T cells in vitro . Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells. Conclusions: In conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL. Clin Cancer Res; 22(7); 1572–82. ©2015 AACR . See related commentary by Bachireddy and Wu, p. 1547

Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting

Abstract: Background Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing. Methods A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM. Results/Conclusion Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 106 and 5 × 106 bone marrow cells to be measured, respectively. © 2015 International Clinical Cytometry Society

Interactions between Ibrutinib and Anti-CD20 Antibodies: Competing Effects on the Outcome of Combination Therapy.

Abstract: Purpose: Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAb) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in preclinical studies, in vitro ibrutinib was reported to decrease CD20 expression and inhibit cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox. Experimental Design: Patients received single-agent ibrutinib (420 mg daily) on an investigator-initiated phase II trial. Serial blood samples were collected pretreatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs. Results: We demonstrate that CD20 expression on ibrutinib was rapidly and persistently downregulated (median reduction 74%, day 28, P < 0.001) compared with baseline. Concomitantly, CD20 mRNA was decreased concurrent with reduced NF-κB signaling. An NF-κB binding site in the promoter of MS4A1 (encoding CD20) and downregulation of CD20 by NF-κB inhibitors support a direct transcriptional effect. Ex vivo , tumor cells from patients on ibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pretreatment cells (median reduction 75%, P < 0.001); however, opsonization by the complement protein C3d, which targets cells for phagocytosis, was relatively maintained. Expression of decay-accelerating factor (CD55) decreased on ibrutinib, providing a likely mechanism for the preserved C3d opsonization. In addition, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy. Conclusions: Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal antitumor effects of such combinations requires further investigation. Clin Cancer Res; 1–10. ©2015 AACR.

Myeloma minimal residual disease testing in the United States: Evidence of improved standardization.

Abstract: activity in DBA patients due to mutations in GATA-1 and without RP mutations. In this subset, eADA activity also was normal suggesting that elevated eADA is a specific feature of DBA associated with RP mutations. It is noteworthy that patients with RPS19 mutations, who tend to have a milder clinical phenotype with fewer congenital anomalies, had lower (but still elevated) levels of eADA when compared to other RP mutations, in particular RPL11 and RPL5. In our cohort, the difference in mean eADA between RPS19 and RPL11 was statistically significant (P 5 0.0150) and a trend toward RPL5 (P 5 0.1264). DBA patients with mutations in RPL11 and RPL5 have been shown to have a higher association with physical malformations [6]. A larger number of patient samples in future studies should improve the statistical power to compare eADA levels between individual RP mutations. This is the first study to assess the value of eADA in another disorder of ribosomal haploinsufficiency [del(5q) MDS] and in DBA associated with GATA-1 mutations . In summary, we found that an elevated eADA strongly suggests the diagnosis of DBA although a normal eADA does not exclude the diagnosis, particularly in the setting of GATA-1 mutations. There does not appear to be a utility for using eADA in the diagnosis of the 5q2 syndrome despite the connection of the disease with impaired ribosome function. The reason for elevated eADA activity in DBA with RP mutations remains to be determined.

A bright and colorful future for DNA cell cycle analysis.

Abstract: DNA cell cycle analysis/DNA content analysis (DCA) by flow cytometry is an important tool that has existed for decades. In pathology, DCA is informative in the diagnosis of partial versus complete molar pregnancy, and has provided prognostically relevant information in the evaluation of breast, prostatic and bladder carcinoma. In the field of Hematopathology, DCA has also yielded important prognostic information. Historically, S phase fraction (SPF) was shown to be an independent prognostic variable in non-Hodgkin lymphoma (1,2). SPF may be used to differentiate indolent from aggressive lymphomas, and complement morphologic/immunohistochemistry studies, such as Ki-67 or mitotic index, in assessing cellular proliferation. Chromosomal aberrations are a common feature of hematolymphoid neoplasms. While DCA by flow cytometry may not detect cytogenetic aberrations in some diseases (e.g., acute myeloid leukemia), where balanced translocations or duplications of smaller chromosomes minimally alter DNA content, there are many hematolymphoid malignancies, such as lymphoblastic leukemia, plasma cell myeloma, and nonhodgkin lymphoma, where abnormalities in DNA ploidy are frequently detected. DNA ploidy is an important prognostic feature in several hematolymphoid malignancies, such as B-lymphoblastic leukemia and plasma cell myeloma (PCM). In B-lymphoblastic leukemia (B-ALL), the presence of hyperdiploidy has been shown to be associated with better prognosis, while poorer outcomes were associated with hypodiploidy (3). PCM is divided into hyperdiploid (more favorable prognosis) and non-hyperdiploid (less favorable prognosis) subtypes, and the demonstration of hypodiploidy portends a poor prognosis (4). Despite the valuable information that DCA provides, some limitations have prevented it from being fully utilized in the clinical flow cytometry diagnostic laboratory, specifically in Hematopathology diagnosis. Some DNA dyes, such as the Hoechst dyes, required specialized UV lasers that were not standard issue on clinical instruments. Dyes such as propidium iodide, and the DRAQ dyes could be used on clinical instruments, but there were limitations in the number additional surface markers (usually only 3 to 4 markers) that could be used concurrently, due to the bleed-over of signal into adjacent channels (5,6). Ultimately, with the growing need to perform minimal residual disease testing, and the push for better resolution of rare neoplastic cell populations, utilization of all available channels of a clinical flow cytometer for surface marker detection (instead of for DCA) certainly was the most pragmatic approach. In the last decade, however, the modern clinical flow cytometry has undergone an evolution. Clinical flow cytometers boasting capabilities for 8 or more colors are now quite commonplace, whereas several years ago, they were unusual. Some major academic institutions and private laboratories are now even performing 9 and 10 color-flow cytometry, routinely (7). Improved instrumentation with multiple lasers, and the availability of new reagents, including tandem fluorochromes, has made this possible. Additionally, improvements in software and hardware have improved listmode data analysis, such that analyzing a million events or more per tube is now an easily manageable task. The result is that clinical flow cytometry is now able to detect neoplastic cells, at, or almost at, the same level of sensitivity as molecular methods. Having

Introduction to multiple myeloma special issue: The flow cytometric detection of minimal residual disease.

Abstract: Patient survival after a diagnosis of plasma cell myeloma and subsequent therapy has been found to be critically dependant upon the detection of minimal residual disease (MRD) which is best identified by multiparameter flow cytometry. The flow cytometric measurement of MRD has been determined to be prognostic and a bellwether of a patient’s response to therapy. The need for the practical guidelines presented in this special issue of Clinical Cytometry, based on the work of internationally recognized experts in the field, focuses specifically on myeloma detection. It is hoped that this will help address, clarify and bring consensus to many of the flow cytometric problems we face in the laboratory related to the identification of this disease. I would like to personally thank all contributing authors from both the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis for their combined efforts and contributions to this Special Issue of Clinical Cytometry. Extra-special thanks and acknowledgement goes to our team of superb Guest Editors (C. Yuan, P. Lin, M. Stetler-Stevenson and G. Marti) for their incredible devotion to bringing this very important issue of our Journal to us. Additionally, I would like to acknowledge C. Yuan for her help with the creative cover art and collection of the individual author photos. Critically important was the help and cooperation of our Publisher, John Wiley (T. Manbeck & M. Ricci) and the superb organizational wizardry of our Managing Editor, Mrs. D. Regal.

Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis

Abstract: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a neoplasm of mature B lymphocytes involving peripheral blood (PB), bone marrow (BM), spleen, and lymph nodes (LN). CLL is the most common lymphoproliferative disorder in western countries and is primarily a disease of adults, often occurring during or after middle age. The diagnosis is established by blood counts, blood smears, and immunophenotyping by flow cytometry (FC) of circulating B lymphocytes (1). Although usually an indolent disease, some patients have a more rapid disease progression and require treatment earlier. Survival in patients varies from 1 year to 20 years with an 82% 5-year survival rate (2). FC demonstration of the typical CLL immunophenotype is vital for diagnosis. The differential diagnosis of CLL/SLL would primarily include monoclonal B-cell lymphocytosis (MBL) and mantle cell lymphoma (MCL), although B-prolymphocytic leukemia, marginal zone lymphoma, diffuse large B-cell lymphoma, and lymphoplasmacytic lymphoma can on occasion have some features of CLL/SLL (Table 1) (3).