New directions in chimeric antigen receptor T cell [CAR-T] therapy and related flow cytometry.

Chimeric antigen receptor (CAR) T cells provide a promising approach to the treatment of hematologic malignancies and solid tumors. Flow cytometry is a powerful analytical modality, which plays an expanding role in all stages of CAR T therapy, from lymphocyte collection, to CAR T cell manufacturing, to in vivo monitoring of the infused cells and evaluation of their function in the tumor environment. Therefore, a thorough understanding of the new directions is important for designing and implementing CAR T-related flow cytometry assays in the clinical and investigational settings. However, the speed of new discoveries and the multitude of clinical and preclinical trials make it challenging to keep up to date in this complex field. In this review, we summarize the current state of CAR T therapy, highlight the areas of emergent research, discuss applications of flow cytometry in modern cell therapy, and touch upon several considerations particular to CAR detection and assessing the effectiveness of CAR T therapy.

A phase 1 study of the antibody-drug conjugate brentuximab vedotin with re-induction chemotherapy in patients with CD30-expressing relapsed/refractory acute myeloid leukemia.

BACKGROUND: Outcomes for patients with relapsed/refractory acute myeloid leukemia (R/R AML) remain poor. Novel therapies specifically targeting AML are of high interest. Brentuximab vedotin (BV) is an antibody-drug conjugate that is specific for human CD30. In this phase 1 dose escalation study, the authors evaluated the safety of BV combined with mitoxantrone, etoposide, and cytarabine (MEC) re-induction chemotherapy for patients with CD30-expressing R/R AML. METHODS: Using a standard dose escalation design, the authors evaluated 3 dose levels of BV (0.9 mg/kg, 1.2 mg/kg, and 1.8 mg/kg) administered once on day 1 followed by MEC on days 3 through 7. RESULTS: There were no dose-limiting toxicities noted and the maximum tolerated dose was not reached. The recommended phase 2 dose of BV was determined to be 1.8 mg/kg when combined with MEC. The side effect profile was similar to that expected from MEC chemotherapy alone, with the most common grade ≥3 toxicities being febrile neutropenia, thrombocytopenia, and anemia (toxicities were graded using the National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.0]). Among the 22 patients enrolled on the trial, the composite response rate was 36%, with a composite response rate of 42% noted among those who received the highest dose of BV. The median overall survival was 9.5 months, with a median disease-free survival of 6.8 months observed among responders. Approximately 55% of patients were able to proceed with either allogeneic hematopoietic stem cell transplantation or donor lymphocyte infusion. CONCLUSIONS: The combination of BV with MEC was found to be safe in patients with CD30-expressing R/R AML and warrants further study comparing this combination with the use of MEC alone in this population (ClinicalTrials.gov identifier NCT01830777). LAY SUMMARY: The outcomes for patients with relapsed/refractory acute myeloid leukemia (R/R AML) are exceptionally poor. New and emerging treatment combinations are actively being studied in an effort to improve outcomes. The authors examined the combination of brentuximab vedotin, an antibody product that recognizes a marker called CD30, with mitoxantrone, etoposide, and cytarabine (MEC), a common chemotherapy regimen, in patients with R/R AML that expressed the CD30 marker. The authors found that the combination was safe and well tolerated. Future studies comparing this new combination with the use of MEC alone can help to inform its effectiveness for this patient population.

Anti-CD37 chimeric antigen receptor T cells are active against B- and T-cell lymphomas.

Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

Metal Ion Levels Are Not Correlated With Histopathology of Adverse Local Tissue Reactions in Taper Corrosion of Total Hip Arthroplasty.

BACKGROUND: The underlying biological mechanism in the formation of adverse local tissue reaction in taper corrosion of total hip arthroplasty (THA) remains unknown. This study evaluated whether there was a dose-dependent relationship between metal ion levels, intraoperative tissue damage and ALVAL (aseptic lymphocyte-dominated vasculitis-associated lesion) scores in dual taper THA patients who underwent revisions for taper corrosion. METHODS: We performed a retrospective review of 31 dual taper THA patients who underwent revision surgery from May 2013 to October 2013. Preoperative serum metal ion levels, intraoperative tissue damage grading, and ALVAL scores were reviewed. Multivariate analysis was performed to determine if an association existed between metal ion levels, intraoperative tissue damage, and ALVAL scores. RESULTS: Findings consistent with adverse local tissue reaction were found in all cases. We noted 10 patients with low, 8 with moderate, and 13 with high ALVAL scores, respectively. For intraoperative tissue damage, we recorded 2 (grade 1), 22 (grade 2) and 7 (grade 3) cases. Preoperatively, there was preferential elevation of serum cobalt (3.8 ng/mL, 2.3-17.0) compared to serum chromium (1.0 ng/mL, 0.2-5.8). There was no correlation between preoperative metal ion levels and intraoperative tissue damage (R = -0.06, P = .74) or ALVAL scores (R = -0.04, P = .481). There was also no correlation between intraoperative tissue damage and ALVAL score (R = -0.06, P = .73). CONCLUSION: There was no significant correlation between ALVAL scores and prerevision surgery metal ion levels or intraoperative tissue damage, suggesting that the biological mechanism of histologic morphology cannot be solely attributed to elevated metal ion levels and is likely multifactorial, reflecting a complex interplay between implant and patient factors.

IgG4-related Orbital Disease and Its Mimics in a Western Population.

Although chronic inflammatory disorders of the ocular adnexa are relatively common, their pathogenesis is in many cases poorly understood. Recent investigation suggests that many cases of sclerosing orbital inflammation are a manifestation of IgG4-related disease; however, most patients reported have been Asian, and it is not clear whether the results of studies from the Far East can be reliably extrapolated to draw conclusions about Western patients. We evaluated 38 cases previously diagnosed as orbital inflammatory pseudotumor or chronic dacryoadenitis to determine whether our cases fulfill the criteria for IgG4-RD (IgG4-related dacryoadenitis when involving the lacrimal gland, and IgG4-related sclerosing orbital inflammation when involving orbital soft tissue). Fifteen patients had IgG4-related dacryoadenitis or orbital inflammation. These patients included 9 men and 6 women, aged 24 to 77 years (median, 64 y). Lesions involved orbital soft tissue (8 cases), lacrimal gland (6 cases), and canthus (1 case). In 1 case, focal in situ follicular neoplasia was seen in a background of IgG4-RD. In another case, a clonal IGH gene rearrangement was detected. Four patients with IgG4-RD had evidence of IgG4-RD in other anatomic sites. Five patients, 1 man and 4 women, aged 26 to 74 years (median 50 y) had orbital lesions (2 involving lacrimal gland, 3 involving soft tissue) suspicious for, but not diagnostic of, IgG4-RD. Of 16 patients with IgG4-RD or probable IgG4-RD with information available regarding the course of their disease, 11 patients experienced recurrent or persistent orbital disease. However, no patient developed lymphoma, and no patient died of complications of IgG4-RD. Eighteen patients had lesions not representing IgG4-RD. They included 6 male and 12 female individuals aged 6 to 77 years (median, 47 y). These patients had a variety of diseases, including granulomatosis with polyangiitis (3 cases), Rosai-Dorfman disease (1 case), nonspecific chronic inflammation and fibrosis involving lacrimal gland or soft tissue (12 cases), and others. Clinical and pathologic findings among our patients with IgG4-RD involving the orbit are similar to those previously described in Asian patients. Careful evaluation of histologic and immunophenotypic features and clinical correlation are required to distinguish orbital IgG4-RD from other sclerosing inflammatory lesions in the orbit.

Probability state modeling theory.

As the technology of cytometry matures, there is mounting pressure to address two major issues with data analyses. The first issue is to develop new analysis methods for high-dimensional data that can directly reveal and quantify important characteristics associated with complex cellular biology. The other issue is to replace subjective and inaccurate gating with automated methods that objectively define subpopulations and account for population overlap due to measurement uncertainty. Probability state modeling (PSM) is a technique that addresses both of these issues. The theory and important algorithms associated with PSM are presented along with simple examples and general strategies for autonomous analyses. PSM is leveraged to better understand B-cell ontogeny in bone marrow in a companion Cytometry Part B manuscript. Three short relevant videos are available in the online supporting information for both of these papers. PSM avoids the dimensionality barrier normally associated with high-dimensionality modeling by using broadened quantile functions instead of frequency functions to represent the modulation of cellular epitopes as cells differentiate. Since modeling programs ultimately minimize or maximize one or more objective functions, they are particularly amenable to automation and, therefore, represent a viable alternative to subjective and inaccurate gating approaches.

Human B-cell and progenitor stages as determined by probability state modeling of multidimensional cytometry data.

BACKGROUND: Human progenitor and B-cell development is a highly regulated process characterized by the ordered differential expression of numerous cell-surface and intracytoplasmic antigens. This study investigates the underlying coordination of these modulations by examining a series of normal bone marrow samples with the method of probability state modeling or PSM. RESULTS: The study is divided into two sections. The first section examines B-cell stages subsequent to CD19 up-regulation. The second section assesses an earlier differentiation stage before and including CD19 up-regulation. POST-CD19 ANTIGENIC UP-REGULATION: Statistical analyses of cytometry data derived from sixteen normal bone marrow specimens revealed that B cells have at least three distinct coordinated changes, forming four stages labeled as B1, B2, B3, and B4. At the end of B1; CD34 antigen expression down-regulates with TdT while CD45, CD81, and CD20 slightly up-regulate. At the end of B2, CD45 and CD20 up-regulate. At the end of B3 and beginning of B4; CD10, CD38, and CD81 down-regulate while CD22 and CD44 up-regulate. PRE-CD19 ANTIGENIC UP-REGULATION: Statistical analysis of ten normal bone marrows revealed that there are at least two measurable coordinated changes with progenitors, forming three stages labeled as P1, P2, and P3. At the end of P1, CD38 up-regulates. At the end of P2; CD19, CD10, CD81, CD22, and CD9 up-regulate while CD44 down-regulates slightly. CONCLUSIONS: These objective results yield a clearer immunophenotypic picture of the underlying cellular mechanisms that are operating in these important developmental processes. Also, unambiguously determined stages define what is meant by "normal" B-cell development and may serve as a preliminary step for the development of highly sensitive minimum residual disease detection systems. A companion article is simultaneously being published in Cytometry Part A that will explain in further detail the theory behind PSM. Three short relevant videos are available in the online supporting information for both of these papers.

Diagnostic utility of cerebrospinal fluid flow cytometry in patients with and without prior hematologic malignancy.

Flow cytometry (FCM) is an adjunct study to routine analysis of cerebrospinal fluid (CSF) to investigate for involvement by a hematologic malignancy. However, in our experience, FCM only infrequently detects abnormalities in CSF. To help optimize resources without forfeiting clinically important data, we sought to determine evidence-based indications and criteria for performing FCM on CSF. FCM results of 316 consecutive CSF specimens were retrospectively reviewed and correlated with clinical history, total nucleated cell (TNC) counts, and results of concurrent cytologic review. Of 255 samples adequate for analysis, 54% were from patients with a prior history of hematologic malignancy, of which 12% (17 cases) were abnormal by FCM. Corresponding TNC counts among samples with abnormal FCM ranged from 0-1050 cells/µL, and only 44% showed abnormal morphology on concurrent cytology. Of the remaining 46% of samples from patients with no known history of hematologic malignancy who had CSF sampling for neurological indications, only one (1%) was abnormal by FCM. This specimen had an elevated TNC count (39 cells/µL) but lacked clearly abnormal findings on concurrent cytology. These results support the use of CSF FCM only in patients with a history of hematologic malignancy or, in the absence of such a history, in samples showing pleocytosis. If these criteria were applied to the current cohort using a TNC count cut-off of >5 cells/µL, 23% of samples would have been deferred from testing, resulting in decreased cost, improved efficiency, and reduction in the need for unnecessary testing without a negative impact on clinical care.

Distinct, strict requirements for Gfi-1b in adult bone marrow red cell and platelet generation.

The zinc finger transcriptional repressor Gfi-1b is essential for erythroid and megakaryocytic development in the embryo. Its roles in the maintenance of bone marrow erythropoiesis and thrombopoiesis have not been defined. We investigated Gfi-1b's adult functions using a loxP-flanked Gfi-1b allele in combination with a novel doxycycline-inducible Cre transgene that efficiently mediates recombination in the bone marrow. We reveal strict, lineage-intrinsic requirements for continuous adult Gfi-1b expression at two distinct critical stages of erythropoiesis and megakaryopoiesis. Induced disruption of Gfi-1b was lethal within 3 wk with severely reduced hemoglobin levels and platelet counts. The erythroid lineage was arrested early in bipotential progenitors, which did not give rise to mature erythroid cells in vitro or in vivo. Yet Gfi-1b(-/-) progenitors had initiated the erythroid program as they expressed many lineage-restricted genes, including Klf1/Eklf and Erythropoietin receptor. In contrast, the megakaryocytic lineage developed beyond the progenitor stage in Gfi-1b's absence and was arrested at the promegakaryocyte stage, after nuclear polyploidization, but before cytoplasmic maturation. Genome-wide analyses revealed that Gfi-1b directly regulates a wide spectrum of megakaryocytic and erythroid genes, predominantly repressing their expression. Together our study establishes Gfi-1b as a master transcriptional repressor of adult erythropoiesis and thrombopoiesis.

Phase I study of urate oxidase in the reduction of acute graft-versus-host disease after myeloablative allogeneic stem cell transplantation.

Graft-versus-host disease (GVHD) is a donor T cell driven response against host tissue that can complicate allogeneic hematopoietic stem cell transplantation (HSCT). During acute GVHD, endogenous adjuvants such as uric acid are released by damaged host tissue, activating alloreactive donor T cells. A phase I study was conducted at the Massachusetts General Hospital between 2007 and 2010 to test the hypothesis that reduction of uric acid levels during allogeneic HSCT can modulate the development of acute GVHD. Twenty-one patients with hematologic malignancies in complete remission undergoing myeloablative peripheral blood HSCT received recombinant urate oxidase at .20 mg/kg for 5 consecutive days during conditioning. Results were compared with all patients who underwent allogeneic HSCT at our institution during the same time period who met the same inclusion and exclusion criteria but were not enrolled in the study. The only major adverse event was a case of hemolytic anemia in a patient who had glucose-6-phosphate dehydrogenase deficiency. Primary outcome was the cumulative incidence of grades II to IV acute GVHD, which was significantly decreased in the treatment group in the intention-to-treat analysis (57% [12/21] versus 24% [5/21], P = .036) and in the per-protocol analysis (P = .017). Patients who developed acute GVHD had a higher level of serum uric acid during the pretransplantation period compared with those who did not (P < .001). There was no difference in disease-free or overall survival. Our study suggests that urate oxidase can be safely administered during myeloablative conditioning and may reduce the incidence of acute GVHD.

Teriparatide (PTH 1-34) treatment increases peripheral hematopoietic stem cells in postmenopausal women.

Cells of the osteoblast lineage play an important role in regulating the hematopoietic stem cell (HSC) niche and early B-cell development in animal models, perhaps via parathyroid hormone (PTH)-dependent mechanisms. There are few human clinical studies investigating this phenomenon. We studied the impact of long-term daily teriparatide (PTH 1-34) treatment on cells of the hematopoietic lineage in postmenopausal women. Twenty-three postmenopausal women at high risk of fracture received teriparatide 20 mcg sc daily for 24 months as part of a prospective longitudinal trial. Whole blood measurements were obtained at baseline, 3, 6, 12, and 18 months. Flow cytometry was performed to identify hematopoietic subpopulations, including HSCs (CD34+/CD45(moderate); ISHAGE protocol) and early transitional B cells (CD19+, CD27-, IgD+, CD24[hi], CD38[hi]). Serial measurements of spine and hip bone mineral density (BMD) as well as serum P1NP, osteocalcin, and CTX were also performed. The average age of study subjects was 64 ± 5 years. We found that teriparatide treatment led to an early increase in circulating HSC number of 40% ± 14% (p = 0.004) by month 3, which persisted to month 18 before returning to near baseline by 24 months. There were no significant changes in transitional B cells or total B cells over the course of the study period. In addition, there were no differences in complete blood count profiles as quantified by standard automated flow cytometry. Interestingly, the peak increase in HSC number was inversely associated with increases in bone markers and spine BMD. Daily teriparatide treatment for osteoporosis increases circulating HSCs by 3 to 6 months in postmenopausal women. This may represent a proliferation of marrow HSCs or increased peripheral HSC mobilization. This clinical study establishes the importance of PTH in the regulation of the HSC niche within humans. © 2014 American Society for Bone and Mineral Research.

Flow cytometry for hematopoietic cells.

Within the last 25 years, flow cytometry and fluorescence-activated cell sorting have emerged as both routine diagnostic tools in clinical medicine and as advanced analytic tools critical in performing scientific research. This chapter aims at summarizing the use of flow cytometry in benign and malignant hematology and the monitoring of inherited and acquired immunodeficiency states. Numerous figures are provided from our laboratories at Massachusetts General Hospital that illustrate examples of these conditions. The chapter also describes novel flow cytometry-based imaging techniques, the combination of flow cytometry and mass spectrography, new software tools, and some future directions and applications of advanced instrumentation for flow cytometry.

Flow cytometry is of limited utility in the early identification of "double-hit" B-cell lymphomas.

BACKGROUND: B-cell lymphomas with concurrent translocations of MYC and BCL2 or BCL6, also known as "double-hit" lymphomas (DHL), are rare malignancies characterized by aggressive clinical behavior and poor prognosis. Previous reports suggest that decreased CD20 and/or CD19 expression by flow cytometry is relatively common in DHL and may help to identify cases requiring additional cytogenetic analysis. METHODS: We conducted a retrospective analysis of 26 cases of DHL, and compared their flow cytometric characteristics to cases of Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). Cases were analyzed by four-color flow cytometry, and bivariate dot-plots were reviewed for light scatter characteristics, CD19, CD20, CD45, and surface light chain. RESULTS: Relatively few DHL cases showed dim expression of CD19 or CD20, and statistically significant differences were found only in the frequency of dim CD19 expression between DHL and BL or DLBCL. Although concomitant dim CD19 and CD20 expression was exclusive to DHL, it was present in only a minority of cases. CONCLUSIONS: We conclude that although a subset of DHL expresses aberrant levels of CD19 and/or CD20 by flow cytometry, these findings are of limited utility in identifying cases requiring cytogenetic analysis due to their low frequency. Until more sensitive pathologic parameters can be identified and validated, the decision to perform cytogenetic analysis should rest on a combination of clinical, morphologic, and immunophenotypic features suggestive of high-grade, aggressive disease.

The hematopoietic stem cell niche--home for friend and foe?

The hematopoietic stem cell (HSC) niche is involved in the maintainance and regulation of quiescence, self-renewal and differentiation of hematopoietic stem cells and the fate of their progeny in mammals dealing with the daily stresses to the hematopoietic system. From the discovery that perturbations of the HSC niche can lead to hematopoietic disorders, we have now arrived at the prospect that the HSC niche may play a role in hematological malignancies and that this HSC niche may be a target for therapy. This review attempts to capture the discoveries of the last few years regarding the normal and malignant hematopoietic stem cell niche and possible ways to target this niche.

Relationship between ABO genotype and A antigen expression on platelets.

BACKGROUND: Although platelets (PLTs) are known to express ABH antigens, the extent of expression is different on PLTs compared with red blood cells and the relationship between PLT ABH expression and genotype has not been thoroughly investigated. STUDY DESIGN AND METHODS: We measured blood group H and A antigens on PLTs from 100 normal volunteers using fluorescent-conjugated reagents and flow cytometry. Individuals were also genotyped at the ABO locus using a commercially available genotyping system. RESULTS: Expression of A and H antigen varied widely on PLTs from different individuals. Among group A and AB persons, H antigen expression was significantly greater than A antigen (p < 0.0001). The ratio of H-to-A antigen expression varied more than 100-fold in a predictable fashion according to genotype with values lowest in A1/A1 < A1/O < A2/A2 < A2/O. H and A antigen expression was unaffected by secretor status. The proportion of PLTs with high A expression also varied directly according to genotype. CONCLUSIONS: Blood group A and H antigen expression on PLTs varies in a predictable fashion according to genotype. Flow cytometry and genotyping identify individuals who strongly express A antigens, a finding that may be relevant to clinical PLT transfusion across ABO barriers.