Dalia A. Salem

Partial reconstitution of humoral immunity and fewer infections in patients with chronic lymphocytic leukemia treated with ibrutinib.

Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia. Therefore, we assessed the impact of ibrutinib on immunoglobulin levels, normal B cells, and infection rate in patients with CLL treated with single-agent ibrutinib on a phase 2 investigator-initiated trial. Consistent with previous reports, immunoglobulin G (IgG) levels remained stable during the first 6 months on treatment, but decreased thereafter. In contrast, there were a transient increase in IgM and a sustained increase in IgA (median increase 45% at 12 months, P < .0001). To distinguish the effects on clonal B cells from normal B cells, we measured serum free light chains (FLCs). In κ-clonal CLL cases, clonal (κ) FLCs were elevated at baseline and normalized by 6 months. Nonclonal (λ) FLCs, which were often depressed at baseline, increased, suggesting the recovery of normal B cells. Consistently, we observed normal B-cell precursors in the bone marrow and an increase in normal B-cell numbers in the peripheral blood. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥50% from baseline to 12 months, had a significantly lower rate of infections (P = .03). These data indicate that ibrutinib allows for a clinically meaningful recovery of humoral immune function in patients with CLL. This trial was registered at www.clinicaltrials.gov as #NCT015007330.

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

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; 22(1); 86–95. ©2015 AACR.

Protective effect of adipose-derived mesenchymal stem cells against acute kidney injury induced by ischemia-reperfusion in Sprague-Dawley rats

Acute kidney injury (AKI) is a complex clinical condition associated with significant morbidity and mortality and lacking effective management. Ischemia-reperfusion injury (IRI) remains one of the leading causes of AKI in native and transplanted kidneys. The aim of this study was to evaluate the efficacy of adipose-derived mesenchymal stem cells (ADSCs) in the prevention of renal IRI in rats. The study was conducted on male Sprague-Dawley rats (n=72) weighing 250–300 g. Rats were randomly assigned to three main groups: i) Sham-operated control group (n=24); ii) positive control group, in which rats were subjected to IRI and were administered culture media following 4 h of IRI (n=24); and iii) ADSC group (n=24), in which rats were administered 1×106 ADSCs via the tail vein following 4 h of IRI. Each main group was further divided according to the timing after IRI into four equal-sized subgroups. Renal function was tested via the measurement of serum creatinine levels and creatinine clearance. In addition, malondialdehyde (MDA) levels were determined in serum and renal tissue homogenate as an indicator of oxidative stress. Histopathological changes were analyzed in different regions of the kidney, namely the cortex, outer stripe of the outer medulla (OSOM), inner stripe of the outer medulla (ISOM) and inner medulla. In each region, the scoring system considered active injury changes, regenerative changes and chronic changes. The ADSCs were assessed and their differentiation capability was verified. IRI resulted in a significant increase in serum creatinine, serum and tissue MDA levels and a significant reduction in creatinine clearance compared with those in sham-operated rats,. These changes were attenuated by the use of ADSCs. The prominent histopathological changes in the cortex, ISOM and OSOM were reflected in the injury score, which was significantly evident in the positive control group. The use of ADSCs was associated with significantly lowered injury scores at days 1 and 3; however, no significant effect was observed on day 7. These results indicate that the use of ADSCs ameliorates renal injury and dysfunction associated with IRI in rats.

Prognostic implications of NPM1 mutations and FLT3 internal tandem duplications in Egyptian patients with cytogenetically normal acute myeloid leukemia

Abstract Nucleophosmin (NPM1) and fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) gene mutations represent the most frequent molecular aberrations in patients with cytogenetically normal-acute myeloid leukemia (CN-AML). We analyzed the prognostic impact of these mutations and their interactions in adults with CN-AML. NPM1 mutation (NPM1mut) and FLT3-ITD mutation (FLT3-ITD+) were analyzed by polymerase chain reaction and GeneScan assays of bone marrow samples obtained from newly diagnosed 104 CN-AML patients. FLT3-ITD+ and NPM1mut were detected in 36 (34.6%) and 30 (28.8%) out of 104 subjects, respectively, 16 cases (15.4%) had double NPM1mut/FLT3-ITD+. The incidence of FLT3-ITD+ was significantly higher in the NPM1mut group than in the NPM1 wild (NPM1wt) group (P = 0.018). Statistical analysis revealed that isolated NPM1mut group had a better clinical outcomes in terms of higher complete response (CR) rate (P = 0.01) and a trend towards favorable overall survival (OS) and disease-free survival (DFS) (P = 0.28, 0.40, respectively). In contrast, the isolated FLT3-ITD+ group had an unfavorable outcome in terms of lower CR rate (P = 0.12), shorter OS, and DFS (P < 0.0001 for both). The NPM1mut/FLT3-ITD-group had the best OS and DFS, while the NPM1wt/FLT3-ITD+ group had the worst OS and DFS than other groups (NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD−) (P < 0.0001 for both). Multivariate Cox regression analysis showed that age and FLT3/ITD+ were independent poor prognostic factors for OS (P = 0.006, <0.0001, respectively), while FLT3/ITD+ was independent predictor for DFS (P = 0.04). However, NPM1mut did not have a significant impact on OS and DFS. In conclusion, adult patients with CN-AML carrying isolated NPM1mut and isolated FLT3-ITD+ exhibit different clinical outcomes than those with NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD−. Patients with NPM1mut/FLT3-ITD− had the best prognosis in terms of higher CR, OS, and DFS, while those with NPM1mut/FLT3-ITD+ had the worst CR rate, and NPM1wt/FLT3-ITD+ had the lowest OS and DFS.

Flowcytometric Immunophenotypic Profile of Acute Leukemia: Mansoura Experience

Acute leukemia (AL) displays characteristic patterns of antigen expression, which facilitate their identification and proper classification. The purpose of this study is to evaluate the diagnostic usefulness of commonly used immune-markers for immunophenotyping of AL and to define the best immune-markers to be used for proper diagnosis and classification of AL. Besides, to recognize the frequency of different AL subtypes and the antigen expression profile in our Egyptian patients. We retrospectively analyzed the immunophenotypic data of 164 de novo AL patients from our institution during 2009 and 2010. Among these patients, 68.9% were classified as acute myeloblastic leukemia (AML) while 31.1% classified as acute lymphoblastic leukemia (ALL). The commonest FAB subtype in AML group was AML-M4/5 (34.5%) which may differ from most published data. As regard ALL, there were 74.5% with B-ALL and 25.5% with T-ALL. It was found that combined use of HLADR and CD34 was much more helpful in distinguishing APL from non-APL AML than either of these antigens alone. It was found that cCD79a and CD19 were the most sensitive marker for B-ALL while cCD3, CD7 and CD5 were the most sensitive antigens for T-ALL. Our analysis of AL phenotypes proved that employed antibody panels are adequate for proper diagnosis and classification of AL. Flowcytometry was found to be especially useful in the identification of AML-M0 and differentiation of APL from non-APL AML. Immunophenotyping results and FAB classification of our AL patients were comparable to internationally published studies apart from predominance of AML-M4/5 and more frequent APL.

T Cells Genetically Modified to Express an Anti–B-Cell Maturation Antigen Chimeric Antigen Receptor Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma

Purpose Therapies with novel mechanisms of action are needed for multiple myeloma (MM). T cells can be genetically modified to express chimeric antigen receptors (CARs), which are artificial proteins that target T cells to antigens. B-cell maturation antigen (BCMA) is expressed by normal and malignant plasma cells but not normal essential cells. We conducted the first-in-humans clinical trial, to our knowledge, of T cells expressing a CAR targeting BCMA (CAR-BCMA). Patients and Methods Sixteen patients received 9 × 106 CAR-BCMA T cells/kg at the highest dose level of the trial; we are reporting results of these 16 patients. The patients had a median of 9.5 prior lines of MM therapy. Sixty-three percent of patients had MM refractory to the last treatment regimen before protocol enrollment. T cells were transduced with a γ-retroviral vector encoding CAR-BCMA. Patients received CAR-BCMA T cells after a conditioning chemotherapy regimen of cyclophosphamide and fludarabine. Results The overall response rate was 81%, with 63% very good partial response or complete response. Median event-free survival was 31 weeks. Responses included eradication of extensive bone marrow myeloma and resolution of soft-tissue plasmacytomas. All 11 patients who obtained an anti-MM response of partial response or better and had MM evaluable for minimal residual disease obtained bone marrow minimal residual disease-negative status. High peak blood CAR+ cell levels were associated with anti-MM responses. Cytokine-release syndrome toxicities were severe in some cases but were reversible. Blood CAR-BCMA T cells were predominantly highly differentiated CD8+ T cells 6 to 9 days after infusion. BCMA antigen loss from MM was observed. Conclusion CAR-BCMA T cells had substantial activity against heavily treated relapsed/refractory MM. Our results should encourage additional development of CAR T-cell therapies for MM.

Expression of the IL-6 receptor alpha-chain (CD126) in normal and abnormal plasma cells in monoclonal gammopathy of undetermined significance and smoldering myeloma

Abstract IL-6 activity in normal plasma cells (nPCs) and abnormal plasma cells (aPCs) is CD126 (subunit of IL-6 receptor) dependent. We quantified CD126 expression on nPCs and aPCs in monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), and multiple myeloma (MM). CD126 was detected on all nPCs and aPCs indicating that CD126 does not have diagnostic utility. CD126 expression was higher in aPCs than in nPCs in 85% SMM but only 41% MGUS and there was evidence that CD126 was higher in aPCs than nPCs in the SMM (p = .048) but not MGUS (p = .96) patients. There is also a greater association between nPC and aPC CD126 expression in low risk MGUS than observed in high risk MGUS and SMM, suggesting normal regulation of CD126 decreases with disease progression. Future studies need to elucidate the role of bone marrow milieu versus escape from normal CD126 regulation in malignant transformation of clonal plasma cells.

Quantification of B-cell maturation antigen, a target for novel chimeric antigen receptor T-cell therapy in Myeloma

B-cell maturation antigen (BCMA) is expressed by normal and malignant plasma cells and is targeted via anti-BCMA chimeric antigen receptor T-cell therapy (BCMA CAR T-cell therapy) in plasma cell myeloma (PCM) patients. Surface BCMA expression is required for CAR T-cell binding and killing. We determined the incidence and intensity of expression of BCMA in bone marrow PCM cells using flow cytometry (FC) and immunohistochemistry (IHC). PCM BCMA expression was assessed by FC in 70 patients and in 43 concurrent specimens by IHC. BCMA expression was detected in 94% of patients. FC could assess BCMA expression in all specimens and expression was quantifiable (QuantiBRITE system, BD Biosciences, San Jose, CA) in 89% of cases. Expression was highly variable and could be numerically classified into dim, moderate or bright levels of expression. In the 43 specimens assessed successfully by both IHC and FC, FC showed higher positivity rate (97%) than IHC (72%), indicating that FC is more useful than IHC in detection of BCMA (p = 0.002; McNemars test). We conclude that FC is more sensitive than IHC and can be used to objectively quantify BCMA expression by myeloma cells. IHC is primarily useful when there is significant infiltration of the bone marrow by myeloma and is less sensitive with low numbers of myeloma cells. Furthermore, the ability of FC to differentiate between normal and abnormal plasma cells and to quantify BCMA on these cells, makes it a useful and sensitive tool in screening patients for CAR T-cell therapy and for follow-up post therapy.

Clinical relevance of thrombospondin receptor (CD36) expression in Egyptian de novo adult acute myeloid leukemia

Background Acute myeloid leukemias (AMLs) are a heterogeneous group of disorders that often present with different morphological, immunophenotypic, and cytogenetic patterns. Identification of these characteristics may be useful for a better prognostic evaluation and for a more appropriate therapeutic approach. CD36 is a transmembrane, highly glycosylated, glycoprotein commonly expressed on blasts in acute monocytic leukemia, megakaryoblastic leukemia, and erythroleukemia. Patients and methods We evaluated CD36 surface expression in 97 newly diagnosed AML patients, and the results were correlated with the morphology, immunophenotype, cytogenetic pattern, and clinical outcome. Results CD36 antigen was recorded in 48 of 97 patients (49.5%) and particularly in those with M5 and M6 FAB subtypes. Moreover, CD36 expression was significantly associated with the expression of CD11b ( P =0.001) and CD14 ( P =0.0001), unfavorable cytogenetic abnormalities ( P =0.001), shorter overall survival ( P >0.0001), and leukemia-free survival ( P =0.03). Conclusion On the basis of the results of the study, it can be concluded that CD36 expression in AML patients may identify a subgroup with a poor prognosis, and may thus be a valuable adjunct to be added to the current prognostic factors.

Diagnostic value of T-cell receptor γ gene rearrangement in T-cell neoplasms

Background The discrimination between reactive and malignant cell populations in some patients with suspected T-cell lymphoproliferative disorders can be complicated and less straightforward. Often, the dilemma lies in determining whether a population of lymphocytes is reactive or neoplastic. In such cases, T-cell receptor (TCR) gene clonality studies have proved useful as an additional diagnostic tool. The TCRγ gene is a preferred target for TCR gene clonality as it is rearranged at an early stage of T lymphoid development, rearranged in a large percent of T-cell neoplasms, and also because of its relative structural simplicity. Materials and methods In the present study, we used the BIOMED-2 multiplex primer panel to assess the value of TCRγ gene rearrangement using genescan analysis in 30 patients with suspected T-cell neoplasms and to elucidate its possible role in the diagnosis of such disorders. Results TCRγ gene clonality was detected in all 18 patients with suspected T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma. In contrast, it was detected in nine out of 12 patients (75%) with suspected peripheral T-cell lymphoma. It is known that TCRγ monoclonality in peripheral T-cell lymphoma can be detected in patients with more aggressive disease in terms of both clinical presentation and laboratory results. VγfI was the most frequently used Vγ segment in our patients. The sensitivity and specificity of genescan were found to be 0.89 and 0.67, respectively, as compared with histopathology. Conclusion It is concluded that TCRγ gene clonality is a very useful diagnostic tool in T-cell neoplasms. As it is relatively simple, it can be used as a preliminary test for clonality assessment, followed by the more complex TCRβ gene rearrangement only in negative cases to improve sensitivity. In addition, it should be used together with heteroduplex and interpreted within the clinical context to improve its specificity.