Chimeric antigen receptor (CAR) T-cells targeting CD19 mediate potent effects in relapsed/refractory pre-B cell acute lymphoblastic leukemia (B-ALL) but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed on most B-ALL and usually retained following CD19 loss. We report results from a phase I trial testing a novel CD22-CAR in twenty-one children and adults, including 17 previously treated with CD19-directed immunotherapy. Dose dependent anti-leukemic activity was observed with complete remission in 73% (11/15) of patients receiving ≥ 1 × 106 CD22-CART cells/kg, including 5/5 patients with CD19dim/neg B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted escape from killing by CD22-CART cells. These results are the first to eastablish the clinical activity of a CD22-CAR in pre-B cell ALL, including in leukemia resistant to anti-CD19 immunotherapy, demonstrating comparable potency to CD19-CART at biologically active doses in B-ALL. They also highlight the critical role played by antigen density in regulating CAR function. (Funded by NCI Intramural Research Program)

Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells targeting the CD19 B lineage receptor has demonstrated marked success in relapsed pre-B-cell acute lymphoblastic leukaemia (ALL). Persisting CAR-T cells generate sustained pressure against CD19 that may drive unique mechanisms of resistance. Pre-B ALL originates from a committed pre-B cell or an earlier progenitor, with potential to reprogram into other hematopoietic lineages. Here we report changes in lineage markers including myeloid conversion in patients following CD19 CAR therapy. Using murine ALL models we study the long-term effects of CD19 CAR-T cells and demonstrate partial or complete lineage switch as a consistent mechanism of CAR resistance depending on the underlying genetic oncogenic driver. Deletion of Pax5 or Ebf1 recapitulates lineage reprogramming occurring during CD19 CAR pressure. Our findings establish lineage switch as a mechanism of CAR resistance exposing inherent plasticity in genetic subtypes of pre-B-cell ALL.

Despite high remission rates following CAR-T cell therapy in B-ALL, relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may reduce the likelihood of antigen loss, thus improving sustained remission rates. A systematic approach to the generation of CAR constructs incorporating two target-binding domains led to several novel CD19/CD22 bivalent CAR constructs. Importantly, we demonstrate the challenges associated with the construction of a bivalent CAR format that preserves bifunctionality against both CD19 and CD22. Using the most active bivalent CAR constructs, we found similar transduction efficiency compared to that of either CD19 or CD22 single CARs alone. When expressed on human T cells, the optimized CD19/CD22 CAR construct induced comparable interferon γ and interleukin-2 in vitro compared to single CARs against dual-antigen-expressing as well as single-antigen-expressing cell lines. Finally, the T cells expressing CD19/CD22 CAR eradicated ALL cell line xenografts and patient-derived xenografts (PDX), including a PDX generated from a patient with CD19− relapse following CD19-directed CAR therapy. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce risk of antigen loss.

Determination of the expression and spatial distribution of molecular epitopes, or antigens, in patient tissue specimens has substantially improved the pathologist's ability to classify disease processes. Certain disease pathophysiologies are marked by characteristic increased or decreased expression of developmentally controlled antigens, defined as Cluster of Differentiation markers, that currently form the foundation for understanding lymphoid malignancies. While chromogens and organic fluorophores have been utilitized for some time in immunohistochemical analyses, developments in synthetic, inorganic fluorophore semiconductors, namely quantum dots, offer a versatile alternative reporter system. Quantum dots are stable fluorophores, are resistant to photobleaching, and are attributed with wide excitation ranges and narrow emission spectra. To date, routinely processed, formalin-fixed tissues have only been probed with two quantum dot reporters simultaneously. In the present study, streptavidin-conjugated quantum dots with distinct emission spectra were tested for their utility in identifying a variety of differentially expressed antigens (surface, cytoplasmic, and nuclear). Slides were analyzed using confocal laser scanning microscopy, which enabled with a single excitation wavelength (488 nm argon laser) the detection of up to seven signals (streptavidin-conjugated quantum dots 525, 565, 585, 605, 655, 705 and 805 nm) plus the detection of 4'6-DiAmidino-2-PhenylIndole with an infra-red laser tuned to 760 nm for two photon excitation. Each of these signals was specific for the intended morphologic immunohistochemical target. In addition, five of the seven streptavidin-conjugated quantum dots tested (not streptavidin-conjugated quantum dots 585 or 805 nm) were used on the same tissue section and could be analyzed simultaneously on routinely processed formalin-fixed, paraffin-embedded sections. Application of this multiplexing method will enable investigators to explore the clinically relevant multidimensional cellular interactions that underlie diseases, simultaneously. Keywords: immunofluorescence; quantum dots; immunohistochemistry; multiplexing; multispectral; confocal microscopy For some time, the application of antibodies in the immunohistochemical staining of tissues has markedly improved the classification and diagnosis of disease processes. Nowhere is this more evident than in the lymphoid malignancies, wherein the identification of differentiation antigens, also called CD markers, has fundamentally improved classification of diseases and elucidated the underlying pathophysiology. A constant infusion of new technologies into pathology practice has enabled these changes, including monoclonal antibodies, streptavidin/biotin interactions, antigen retrieval, organic fluorophores, and enzymatic amplification strategies. A novel nanotechnology, the quantum dot, an inorganic fluorophore, promises to offer the next technological breakthrough in the imaging of patient tissues. Quantum dots have recently been ci...

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a rare B-cell lymphoma considered to be of germinal center (GC) derivation. Studies on immunoglobulin expression have been few, and post-switch immunoglobulin (IgG) has been identified in the majority of cases examined thus far. We reviewed 180 cases of NLPHL and observed the unexpected expression of IgD in 27% of cases. IgD is usually coexpressed with IgM in naive B cells but can also be seen as IgD-only in centroblasts (CD38-positive) or memory B cells (CD27-positive). We asked whether IgD-positive NLPHL differed from cases of NLPHL negative for IgD. Clinically, the IgD-positive cases presented at a younger median age (21 vs. 44 years) and had a striking male predominance (male-to-female ratio, 23:1 vs. 1.5:1). Cervical lymph nodes were more frequently involved (56% vs. 18.2%). L&H cells were localized in a predominantly extrafollicular distribution in the majority of IgD-positive cases (69%). The IgD-positive cases did not coexpress IgM or CD27 (a marker associated with memory B cells), and nearly all (93%) were weakly positive for CD38, supporting a GC derivation. The expression of Bcl-6, BOB.1, Oct2, and SWAP-70 was similar in the two groups. However, PU.1 expression was seen in 60% of the IgD-positive cases in contrast to 86% of the IgD-negative cases. The absence of PU.1 staining correlated with more L&H cells in an extrafollicular distribution, weakening the use of this marker in the differential diagnosis with T-cell rich/histiocyte rich B-cell lymphomas. To study IgD expression in "de-novo" T-cell rich/histiocyte rich B-cell lymphomas, we analyzed 20 cases and all but one were negative. In conclusion, cases of IgD-positive NLPHL do not differ from IgD-negative cases regarding cellular derivation and most other immunophenotypic characteristics. However, IgD-positive NLPHL exhibits distinctive clinical features, and more often involves the interfollicular region in a background relatively rich in T cells. IgD positivity may represent an additional useful marker in the diagnosis of NLPHL.

Spindle cell lipoma (SCL), dermatofibrosarcoma protuberans (DFSP), and solitary fibrous tumors (SFT) are cutaneous CD34+ spindle cell tumors that may exhibit histopathologic and immunophenotypic overlap. We sought ways to reliably distinguish among these lesions even in small or superficial biopsies. Ten morphologic characteristics were analyzed in a group of 5 SCLs, 6 cutaneous SFTs, and 12 DFSPs. SFT and DFSP exhibited extensive histopathologic overlap in small or partial biopsies. However, adnexal entrapment, defined as diffuse proliferation of tumor cells around pilosebaceous and eccrine structures with minimal disruption or expansion of the dermis, was a feature seen in 10 of the 12 DFSPs and in none of the SFTs or SCLs. Even when only superficial portions of a lesion were present, this feature was identifiable. Spindle cell lipomas posed little diagnostic difficulty, in part because excisional biopsies were performed in all cases of SCL. The number of samples included in the study is relatively small, in part due to the rarity of cutaneous solitary fibrous tumors. We conclude that careful attention to these histopathologic features enables reliable distinction among these tumors.

To assess the role of interferon regulatory factor (IRF) 8 in B-cell development and lymphomagenesis, we studied its expression in reactive lymphoid tissues, its relationship to other B-cell transcription factors, and its expression in a series of 232 B-cell tumors and 30 cell lines representing a variety of B-cell developmental stages. We found that although IRF8 was detectable in most reactive B-cells, its expression levels differed with developmental stage. Germinal center B cells contained the highest levels of IRF8, with lower levels seen in mantle and marginal zone B cells and none in plasma cells. IRF8 was coexpressed with PAX-5, Pu.1, and B-cell lymphoma (BCL)-6, and similar to BCL-6, was absent from the small population of IRF4-positive germinal center B cells thought to be committed to postgerminal center developmental programs. Similarly, IRF8 was most strongly expressed in lymphomas of germinal center origin with lower levels present in mantle cell lymphomas, chronic lymphocytic leukemia, and marginal zone lymphomas, and no expression observed in plasmacytic/plasmablastic neoplasms. The reciprocal expression pattern with IRF4 in reactive tissues was generally maintained in lymphomas with some exceptions. These results suggest an important role for IRF8 during germinal center B-cell development and in related lymphomas, and provide a new diagnostic marker helpful in distinguishing B-cell non-Hodgkin lymphoma subtypes.

Cdc7 expression differs significantly among cutaneous melanocytic neoplasms and can be evaluated by routine immunohistochemical methods. The results suggest that differences in Cdc7 expression may account for some of the differences between malignant melanomas and benign melanocytic nevi.