Pre-existing Thyroiditis Ameliorates Papillary Thyroid Cancer: Insights From a New Mouse Model.

Papillary thyroid cancer (PTC) often co-occurs with Hashimoto's thyroiditis, an association that has long been reported in clinical studies yet remains controversial. Some studies, in fact, have suggested a protective effect of thyroiditis while others have not. We generated a mouse model where PTC and thyroiditis develop in a predictable manner, combining the oncogenic drive of the BRAFv600E mutation (inducible by tamoxifen) to the thyroiditis susceptibility of the NOD.H2h4 strain (inducible by iodine). A total of 113 NOD.H2h4_TPO-CRE-ER_BRAFV600E mice (50 followed throughout lifetime and 63 sacrificed at 16 weeks post tamoxifen) were used to determine whether the PTC phenotype differs when thyroiditis precedes or coincides with the onset of PTC. Mice with pre-existing thyroiditis lived longer (median survival of 28.2 weeks post tamoxifen) than those with concomitant (25.6 weeks) or no (24.5 weeks) thyroiditis (P < 0.01 by Laplace regression). PTC developed less frequently (33%) in the pre-existing thyroiditis group than the concomitant (100%) or no (100%) thyroiditis groups (P < 0.001 by chi-squared) and showed less aggressive histopathological features. The intratumoral mononuclear cell infiltration was more prominent in mice with pre-existing thyroiditis (P = 0.002 vs the other groups) and sustained by a significant expansion of effector memory CD8 + T cells and CD19 + B cells. These findings shed light on the controversial PTC-thyroiditis association and emphasize the contribution of intratumoral T and B lymphocytes to the evolution of PTC.

Woodchuck Hepatitis Virus Post-Transcriptional Regulation Element (WPRE) Promotes Anti-CD19 BiTE Expression in Expi293 Cells.

Background: Bispecific antibodies represent an important class of monoclonal antibodies (mAbs), with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti-CD3 × CD19 bispecific antibody (bsAb) is a bispecific T-cell engager currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods: Woodchuck hepatitis virus post-transcriptional regulation (WPRE) sequence was incorporated at the 3' end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion: Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in human embryonic kidney-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody.

Extracellular Vesicle Surface Signatures in IPF Patients: A Multiplex Bead-Based Flow Cytometry Approach.

Background: Extracellular vesicles (EVs) are secreted by cells from their membrane within circulation and body fluids. Knowledge of the involvement of EVs in pathogenesis of lung diseases is increasing. The present study aimed to evaluate the expression of exosomal surface epitopes in a cohort of idiopathic pulmonary fibrosis (IPF) patients followed in two Italian Referral Centres for Interstitial Lung Diseases, comparing them with a group of healthy volunteers. Materials and Methods: Ninety IPF patients (median age and interquartile range (IQR) 71 (66-75) years; 69 males) were selected retrospectively. Blood samples were obtained from patients before starting antifibrotic therapy. A MACSPlex Exosome Kit, human, (Miltenyi Biotec, Bergisch-Gladbach, Germany), to detect 37 exosomal surface epitopes, was used. Results: CD19, CD69, CD8, and CD86 were significantly higher in IPF patients than in controls (p = 0.0023, p = 0.0471, p = 0.0082, and p = 0.0143, respectively). CD42a was lower in IPF subjects than in controls (p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls (p = 0.0007, p = 0.0050, p = 0.0139, and p = 0.0335, respectively). Kaplan-Meier survival analysis for IPF patients: for median values and a cut-off of 0.48 for CD25, the two subgroups showed a significant difference in survival rate (p = 0.0243, hazard ratio: 0.52 (95%CI 0.29-0.92); the same was true for CD8 (cut-off 1.53, p = 0.0309, hazard ratio: 1.39 (95%CI 0.75-2.53). Conclusion: Our multicenter study showed for the first time the expression of surface epitopes on EVs from IPF patients, providing interesting data on the communication signatures/exosomal profile in serum from IPF patients and new insights into the pathogenesis of the disease and a promising reliability in predicting mid-term survival of IPF patients.

Impact of hypoalbuminemia on the prognosis of relapsed/refractory B-cell lymphoma treated with axicabtagene ciloleucel.

INTRODUCTION: Hypoalbuminemia is a known adverse prognostic factor in lymphomas. Yet, it is unknown if axicabtagene ciloleucel (axi-cel) overcomes the adverse prognostic impact of hypoalbuminemia in relapsed/refractory large B-cell lymphoma. METHODS: We conducted a retrospective analysis across three Mayo Clinic centers to assess the relationship of hypoalbuminemia (defined as a serum albumin (SA) levels ≤ 3.5 g/dL) on outcomes of patients treated with axi-cel. RESULTS: This analysis included 81 patients. Two patients had no available SA levels preceding axi-cel infusion. Eighteen patients (22.8%) had hypoalbuminemia with a median SA of 3.3 g/dL. Patients with normal SA had a statistically higher ORR than those without hypoalbuminemia (P = .018). There was no difference in 1-year PFS and OS between the group with hypoalbuminemia and the group with normal SA levels (48% vs 49%, P = .81) and (74% vs 73%, P = .97), respectively. There was no difference in the severity or median duration of cytokine release syndrome or neurotoxicity between the two groups. CONCLUSION: Notwithstanding the limitations related to the relatively small sample size, axi-cel therapy appears to overcome the adverse effect of hypoalbuminemia on OS and PFS. Large multicenter clinical studies are certainly needed to validate these findings.

Tumor-intrinsic and -extrinsic determinants of response to blinatumomab in adults with B-ALL.

Blinatumomab, a bispecific antibody that directs CD3+ T cells to CD19+ tumor cells, shows variable efficacy in B-progenitor acute lymphoblastic leukemia (B-ALL). To determine tumor-intrinsic and -extrinsic determinants of response, we studied 44 adults with relapsed or refractory B-ALL (including 2 minimal residual disease positive) treated with blinatumomab using bulk tumor and single-cell sequencing. The overall response rate in patients with hematological disease was 55%, with a high response rate in those with CRLF2-rearranged Philadelphia chromosome-like ALL (12 [75%] of 16). Pretreatment samples of responders exhibited a tumor-intrinsic transcriptomic signature of heightened immune response. Multiple mechanisms resulted in loss of CD19 expression, including CD19 mutations, CD19-mutant allele-specific expression, low CD19 RNA expression, and mutations in CD19 signaling complex member CD81. Patients with low hypodiploid ALL were prone to CD19- relapse resulting from aneuploidy-mediated loss of the nonmutated CD19 allele. Increased expression of a CD19 isoform with intraexonic splicing of exon 2, CD19 ex2part, at baseline or during therapy was associated with treatment failure. These analyses demonstrate both tumor-intrinsic and -extrinsic factors influence blinatumomab response. We show that CD19 mutations are commonly detected in CD19- relapse during blinatumomab treatment. Identification of the CD19 ex2part splice variant represents a new biomarker predictive of blinatumomab therapy failure.

Changes in Peripheral Blood Neutrophils, Lymphocytes and IL-10 in Children with Kawasaki Disease from Different Age Groups Undergoing Intravenous Immunoglobulin: A Retrospective Study.

Immunoglobulin intravenous (IVIG) is widely used in mucocutaneous lymph node syndrome, known as Kawasaki disease (KD). However, the patients' inflammatory response during usage remains unclear. In the present study, the association between inflammatory response and lymphocyte count in children with KD from different ages was evaluated before and after IVIG. The medical records of 50 children with KD were retrospectively reviewed and divided into five groups according to age. As compared with the data from healthy children, the relative neutrophil count of all children with KD was increased, and that of lymphocytes was decreased. The neutrophil/lymphocyte ratio (NLR) was different among all groups and was higher in children aged ≥4 years, as compared with other groups. Following IVIG, the relative neutrophil and lymphocyte counts of all children with KD returned to normal levels. The altered levels of neutrophils and lymphocytes were found to be linearly correlated. The correlation coefficient in the five groups was 0.99, 0.87, 0.91, 0.97 and 0.99, from young to old, respectively (p < 0.01). The age of children with KD was positively correlated with older age (r = 0.91, p = 0.03). In patients aged ≥4 years, the absolute CD19+ B cell count prior to IVIG increased, and that increase was linearly correlated with the decrease in interleukin-10 (IL-10) following IVIG (r = 0.71, p < 0.05). The older the child's age, the better the regulatory effect of IVIG on the KD child's immune response and the recovery of immune equilibrium it achieved. In KD patients aged ≥4 years, the abnormally proliferating CD19+ B cells may be involved in the secretion of IL-10 to balance the humoral immunity. In such patients, the combination of the absolute CD19+ B cell count prior to IVIG and the decreased levels of IL-10 following IVIG may play a crucial role in evaluating the effect of IVIG in the inflammation.

Biological properties of bone marrow plasma cells influence their recovery in aspirate specimens: impact on classification of plasma cell disorders and potential bias to evaluation of treatment response.

Methods to estimate bone marrow plasma cells (BMPC) basically include histopathology, cytomorphology, and flow cytometry. The present study compares the outcomes of these methods with special focus on the impact of BMPC-specific characteristics on their recovery by either method. Laboratory reports of diagnostic samples from 238 consecutive patients with suspected or known plasma cell disease were retrospectively analyzed. The median (IQR) proportion of BMPC was 30.0% (15.0-70.0%) by histological review (hBMPC), 7.0% (2.0-16.0%) by smear review (sBMPC), and 3.0% (0.8-10.0%) by flow cytometry (fBMPC). The disparity of results between core biopsy and aspirate smear was enhanced in case of poor quality of the smear, increased BM fiber content, higher grade cell atypia, expression of CD56 (all P < 0.0001), the number of cytogenetic aberrations (P = 0.0002), and abnormalities of the MYC gene (P = 0.0002). Conversely, expression of CD19 and a non-clonal plasma cell phenotype were associated with a lower difference between hBMPC and sBMPC (both P < 0.0001). The disparity between the percentages of sBMPC and fBMPC was associated with the quality of the smear (P = 0.0007) and expression of CD56 (P < 0.0001). Our results suggest that the recovery of BMPC in aspirate specimens not only is a matter of sampling quality but also depends on biological cell properties. Aspiration failure due to malignant type features of BMPC may lead to misclassification of plasma cell disorders and represent a bias for the detection of minimal residual disease after therapy.

Negative CD19 expression is associated with inferior relapse-free survival in children with RUNX1-RUNX1T1-positive acute myeloid leukaemia: results from the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-05 study.

We performed a retrospective analysis of leukaemic surface antigen expression and genomic data from a total of 100 RUNX1-RUNX1T1-positive paediatric acute myeloid leukaemia (AML) patients enrolled in the Japanese Paediatric Leukaemia/Lymphoma Study Group (JPLSG) AML-05 protocol to determine risk factors for relapse. In univariate analysis, the KIT exon 17 mutation (n = 21) and CD19 negativity (n = 59) were significant risk factors for relapse (P = 0·01). In multivariate analysis, CD19 negativity was the sole significant risk factor for relapse (hazard ratio, 3·09; 95% confidence interval, 1·26-7·59; P < 0·01), suggesting that biological differences between CD19-positive and CD19-negative RUNX1-RUNX1T1 AML patients should be investigated.

Transitional B cells in quiescent SLE: An early checkpoint imprinted by IFN.

Systemic lupus (SLE) is characterized by a break of B cell tolerance that plays a central role in disease pathophysiology. An early checkpoint defect occurs at the transitional stage leading to the survival of autoreactive B cells and consequently the production of pathogenic autoantibodies. The main purpose of our work was to determine whether transitional B cells, as the most immature naïve B cell subset upstream of pathogenic B cells, display specific features compared to healthy non SLE subjects. Through extensive analysis of transitional B cells from untreated or low treated, mostly Caucasian, SLE patients, we demonstrated that transitional (T1 and T2) B cell frequencies were increased in SLE and positively correlated with disease activity. SLE transitional B cells displayed defects in two closely inter-related molecules (i.e. TLR9 defective responses and CD19 downregulation). RNA sequencing of sorted transitional B cells from untreated patients revealed a predominant overexpression of interferon stimulated genes (ISGs) even out of flares. In addition, early transitional B cells from the bone marrow displayed the highest interferon score, reflecting a B cell interferon burden of central origin. Hence, the IFN signature in transitional B cells is not confined to African American SLE patients and exists in quiescent disease since the medullary stage. These results suggest that in SLE these 3 factors (i.e. IFN imprintment, CD19 downregulation and TLR9 responses impairment) could take part at the early transitional B cell stage in B cell tolerance by-pass, ultimately leading in periphery to the expansion of autoantibodies-secreting cells.

Copy number of the X-linked genes TLR7 and CD40L influences innate and adaptive immune responses.

The number of the X chromosome-linked genes has been previously suggested to influence immune responses and the development of autoimmune diseases. In the present study, we aimed at evaluating the level of expression of CD40L (an X-linked gene involved in adaptive immunity) and TLR7 (an X-linked gene involved in innate immunity) in a variety of different karyotypes. Those included males, females and patients with X chromosome aneuploidy. Healthy females (46, XX; n = 10) and healthy males (46, XY; n = 10) were compared to females with Turner syndrome (TS) (45, X; n = 11) and males with Klinefelter syndrome (KS) (47, XXY; n = 5). Stimulation of peripheral blood mononuclear cells (PBMCs) with PMA and ionomycin resulted in higher percentage of CD3 + CD40L+ T cells (P < 0.001) and higher level expression of CD40L in T cell (P < 0.001) in female and KS patients compared with male and TS patients. TLR7-mediated IFN-alpha production by HLADR + CD3- CD19- cells was significantly upregulated in healthy women compared with healthy males, TS and KS patients (P < 0.001). TLR7 agonist-stimulated PBMCs from healthy females and KS patients expressed significantly higher levels of TLR7 mRNA than those from male and TS patients (P < 0.05). The increased expression of the X-linked genes TLR7 and CD40L in healthy females and KS patients suggests that the presence of two X chromosomes plays a major role in enhancing both innate and adaptive immune responses. These results may contribute to the explanation of sex-based differences in immune biology and the sex bias in predisposition to autoimmune diseases.

GMP CAR-T cell production.

The clinical success achieved using CD19-directed CAR-T cells has stimulated many academic institutions to explore the feasibility of manufacturing these, and other CAR-T cells, in-house. This article reviews the issues that must be addressed in order to achieve this goal. It includes the manufacturing infrastructure, the regulatory environment, practical aspects of production, and the costs involved.

ADCT-402, a PBD dimer-containing antibody drug conjugate targeting CD19-expressing malignancies.

Human CD19 antigen is a 95-kDa type I membrane glycoprotein in the immunoglobulin superfamily whose expression is limited to the various stages of B-cell development and differentiation and is maintained in the majority of B-cell malignancies, including leukemias and non-Hodgkin lymphomas of B-cell origin. Coupled with its differential and favorable expression profile, CD19 has rapid internalization kinetics and is not shed into the circulation, making it an ideal target for the development of antibody-drug conjugates (ADCs) to treat B-cell malignancies. ADCT-402 (loncastuximab tesirine) is a novel CD19-targeted ADC delivering SG3199, a highly cytotoxic DNA minor groove interstrand crosslinking pyrrolobenzodiazepine (PDB) dimer warhead. It showed potent and highly targeted in vitro cytotoxicity in CD19-expressing human cell lines. ADCT-402 was specifically bound, internalized, and trafficked to lysosomes in CD19-expressing cells and, following release of the PBD warhead, resulted in formation of DNA crosslinks that persisted for 36 hours. Bystander killing of CD19- cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent antitumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA crosslinks and γ-H2AX DNA damage response were measured in tumors by 24 hours after single dose administration, whereas matched peripheral blood mononuclear cells showed no evidence of DNA damage. Pharmacokinetic analysis in rat and cynomolgus monkey showed excellent stability and tolerability of ADCT-402 in vivo. Together, these impressive data were used to support the clinical testing of this novel ADC in patients with CD19-expressing B-cell malignancies.

Early Emergence of CD19-Negative Human Antibody-Secreting Cells at the Plasmablast to Plasma Cell Transition.

Long-lived human plasma cells (PCs) play central roles in immunity and autoimmunity and are enriched among the subpopulation of CD19neg human PCs. However, whether human CD19neg PCs are necessarily aged cells that have gradually lost CD19 expression is not known. Assessing peripheral blood samples at steady-state and during the acute response to influenza vaccination in healthy donors, we identify the presence of phenotypic CD19neg plasmablasts, the proliferative precursor state to mature PCs, and demonstrate by ELISPOT that these are Ab-secreting cells (ASCs). During the acute response to influenza vaccination, CD19pos, CD19low, and CD19neg ASCs secrete vaccine-specific Abs and show linked IGHV repertoires. To address precursor/product relationships, we use in vitro models that mimic T-dependent and T-independent differentiation, finding that the CD19neg state can be established at the plasmablast to PC transition, that CD19neg PCs increase as a percentage of surviving PCs in vitro, and that CD19neg and CD19pos PCs can be maintained independently. These data provide proof-of-principle for the view that newly generated ASCs can acquire a mature PC phenotype that is accompanied by loss of CD19 expression at an early stage of differentiation and that aging is not an obligate requirement for a CD19neg state to be established.

Level of PAX5 in differential diagnosis of non-Hodgkin's lymphoma.

BACKGROUND & OBJECTIVES: The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkin's lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis. METHODS: Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, w0 estern blot analysis, and lactate dehydrogenase (LDH) specific staining. RESULTS: B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, Zap700 , HIF 1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients. INTERPRETATION & CONCLUSIONS: The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy.

CMVpp65 Vaccine Enhances the Antitumor Efficacy of Adoptively Transferred CD19-Redirected CMV-Specific T Cells.

PURPOSE: T cells engineered with chimeric antigen receptors (CAR) recognizing CD19 can induce complete remission of B-cell malignancies in clinical trials; however, in some disease settings, CAR therapy confers only modest clinical benefit due to attenuated persistence of CAR T cells. The purpose of this study was to enhance persistence and augment the antitumor activity of adoptively transferred CD19CAR T cells by restimulating CAR(+) T cells through an endogenous cytomegalovirus (CMV)-specific T-cell receptor. EXPERIMENTAL DESIGN: CMV-specific T cells from CMV seropositive healthy donors were selected after stimulation with pp65 protein and transduced with clinical-grade lentivirus expressing the CD19R:CD28:ζ/EGFRt CAR. The resultant bispecific T cells, targeting CMV and CD19, were expanded via CD19 CAR-mediated signals using CD19-expressing cells. RESULTS: The bispecific T cells proliferated vigorously after engagement with either endogenous CMVpp65 T-cell receptors or engineered CD19 CARs, exhibiting specific cytolytic activity and IFNγ secretion. Upon adoptive transfer into immunodeficient mice bearing human lymphomas, the bispecific T cells exhibited proliferative response and enhanced antitumor activity following CMVpp65 peptide vaccine administration. CONCLUSIONS: We have redirected CMV-specific T cells to recognize and lyse tumor cells via CD19CARs, while maintaining their ability to proliferate in response to CMV antigen stimulation. These results illustrate the clinical applications of CMV vaccine to augment the antitumor activity of adoptively transferred CD19CAR T cells in patients with B-cell malignancies.

The role of B cells in the early onset of the first demyelinating event of acute optic neuritis.

PURPOSE: We evaluated the molecular pathways that operate in the early phase of acute optic neuritis (ON) by studying gene expression profiles of peripheral blood mononuclear cells (PBMCs) subpopulations, including CD19(+) B cells, CD14(+) macrophages, and CD4(+) and CD8(+) T cells. METHODS: Samples of PBMC subpopulations were obtained from 18 MS patients within 96 hours of the acute onset of the first demyelinating event of ON, and from 14 age- and sex-matched healthy subjects. High throughput gene expression analysis was performed on samples from six ON patients and nine healthy subjects using Affymetrix technology. Data were analyzed using Partek software, and most informative genes (MIGs) were defined as genes with P < 0.01 and fold change > 2.0. Molecular pathways were analyzed by Ingenuity software. Verification of key MIGs was done on samples from five independent ON patients and five healthy subjects by quantitative (Q) RT-PCR and Western blot. Functional assay to test antigen presentation ability of sorted B cells was performed on blood samples from seven additional ON patients. RESULTS: Significantly differentiating gene expression signatures consisting of 467, 55, and 55 MIGs respective of CD19(+), CD14(+), and CD4(+) cells, were identified between ON and healthy subjects. No MIGs were detected for CD8(+) cells. The major involvement of CD19(+) B cells in the early stage of ON was characterized by enrichment of genes involved in activation of immune mechanisms (P = 3.2 × 10(-25) to 2.5 × 10(-3)), including cellular immune response (P = 7.1 × 10(-12)), B-cell cellular growth and proliferation (P = 1.0 × 10(-7)), activation of immune cells trafficking pathways (P = 5.4 × 10(-15) to 2.0 × 10(-3)), and stimulation of antigen presentation (P = 8.9 × 10(-11)). This massive B-cell-restricted initiation of the immune response in the early disease process of ON was followed by low CD14(+) and CD4(+) cells activity and CD8(+) cells anergy. CONCLUSIONS: Our findings demonstrate that CD19(+) B cells have a significant role in the pathogenesis of the first demyelinating event of acute ON and suggest their role as a possible target for immunomodulation.

Stem cell programs are retained in human leukemic lymphoblasts.

Leukemic lymphoblasts within different immunophenotypic populations possess stem cell properties. However, whether or not the self-renewal program is retained from stem cells or conferred on progenitors by leukemogenic molecules remains unknown. We have addressed the issue in the context of TEL-AML1-associated acute lymphoblastic leukemia (ALL) by profiling a refined program edited from genes essential for self-renewal of hematopoietic stem cells and B-cell development. Bioinformatic analysis shows that ALL populations are loosely clustered and close to the normal population that contains stem and primitive progenitor cells. This finding indicates that immunophenotypes do not reflect maturation stages in ALL and that the self-renewal program may be retained from stem cells. Results of assessing 'first hit' function of TEL-AML1 in different populations of normal cells demonstrate the molecular model. Therefore, the current study shows a leukemogenic scenario of human ALL in which programs of stem cells are sustained in distinct fractions by leukemogenic mutations.

Apparent CD19 expression by natural killer cells: a potential confounder for minimal residual disease detection by flow cytometry in B lymphoblastic leukemia.

Detection of minimal residual disease (MRD) by flow cytometry (FCM) in B lymphoblastic leukemia (B ALL) is important for guiding patient-specific clinical management. We describe apparent expression of CD19 by natural killer cells as a potential confounder in the detection of B ALL MRD by FCM. This finding was noted in seven different patient samples analyzed on different days as part of routine clinical care in our laboratory, with analysis of different anti-CD19 antibody clones and fluorochrome conjugates in five of the seven samples. Although the etiology of this finding is not clear, possibilities include true low level expression and trogocytosis. We highlight this finding to avoid potential misinterpretation when evaluating samples for MRD in patients with B lineage neoplasms, particularly in B ALL.