Adam Bagg

Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia

We designed a lentiviral vector expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19, coupled with CD137 (a costimulatory receptor in T cells [4-1BB]) and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. A low dose (approximately 1.5×10(5) cells per kilogram of body weight) of autologous chimeric antigen receptor-modified T cells reinfused into a patient with refractory chronic lymphocytic leukemia (CLL) expanded to a level that was more than 1000 times as high as the initial engraftment level in vivo, with delayed development of the tumor lysis syndrome and with complete remission. Apart from the tumor lysis syndrome, the only other grade 3/4 toxic effect related to chimeric antigen receptor T cells was lymphopenia. Engineered cells persisted at high levels for 6 months in the blood and bone marrow and continued to express the chimeric antigen receptor. A specific immune response was detected in the bone marrow, accompanied by loss of normal B cells and leukemia cells that express CD19. Remission was ongoing 10 months after treatment. Hypogammaglobulinemia was an expected chronic toxic effect.

T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia

Adoptively transferred gene-modified T cells expand in vivo, eliminate leukemic cells, and form functional memory cells in patients. Go CAR-Ts in the Fast Lane As members of the body’s police force, cells of the immune system vigilantly pursue bad actors that harm healthy tissues, such as infection or cancer, and then try to deter dangerous activity. Researchers have long sought to harness the power of the immune system to fight cancers such as leukemia; however, targeting functional immune T cells to the tumor and maintaining these cells in patients remains challenging. Now, Kalos et al. have genetically modified T cells to express a chimeric antigen receptor (CAR) to yield so-called CAR T cells that specifically target chronic lymphocytic leukemia (CLL) (a B cell cancer). The designer T cells not only expanded, persisted, and attacked tumor cells after transfer into patients; they also mediated cancer remission. Innocent bystanders were also targeted, as reflected by decreased numbers of B cells and plasma cells and the development of hypogammaglobulinemia. The CAR T cells used in this study expressed an antigen receptor that consists of antibody binding domains that bind in a restricted manner to the CD19 protein (which is found solely on normal B cells and plasma cells) attached to both a T cell–specific costimulatory domain and a T cell–specific intracellular signaling domain. The resulting chimeric receptor could activate T cells in response to CD19 in the absence of major histocompatibility complex restriction, allowing for much broader cellular targeting than is obtained with normal T cells. After transfer into three CLL patients, these CAR T cells expanded >1000-fold, persisted for more than 6 months, and eradicated CLL cells. Some of these CAR T cells persisted with a memory phenotype, which would allow them to respond more quickly and on a larger scale with a second exposure to CLL cells. Indeed, two of the three CLL patients who underwent the CAR T cell treatment had complete remission of their leukemia. Although this is early in the clinical study, these results highlight the potential for CAR-modified T cells to bring cancer therapy up to speed. Tumor immunotherapy with T lymphocytes, which can recognize and destroy malignant cells, has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens. Chimeric antigen receptors (CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens; however, to date, lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials. We report that CAR T cells that target CD19 and contain a costimulatory domain from CD137 and the T cell receptor ζ chain have potent non–cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia (CLL). The engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months. Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia. On average, each infused CAR-expressing T cell was calculated to eradicate at least 1000 CLL cells. Furthermore, a CD19-specific immune response was demonstrated in the blood and bone marrow, accompanied by complete remission, in two of three patients. Moreover, a portion of these cells persisted as memory CAR+ T cells and retained anti-CD19 effector functionality, indicating the potential of this major histocompatibility complex–independent approach for the effective treatment of B cell malignancies.

Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma

An obstacle to cancer immunotherapy has been that the affinity of T-cell receptors (TCRs) for antigens expressed in tumors is generally low. We initiated clinical testing of engineered T cells expressing an affinity-enhanced TCR against HLA-A*01-restricted MAGE-A3. Open-label protocols to test the TCRs for patients with myeloma and melanoma were initiated. The first two treated patients developed cardiogenic shock and died within a few days of T-cell infusion, events not predicted by preclinical studies of the high-affinity TCRs. Gross findings at autopsy revealed severe myocardial damage, and histopathological analysis revealed T-cell infiltration. No MAGE-A3 expression was detected in heart autopsy tissues. Robust proliferation of the engineered T cells in vivo was documented in both patients. A beating cardiomyocyte culture generated from induced pluripotent stem cells triggered T-cell killing, which was due to recognition of an unrelated peptide derived from the striated muscle-specific protein titin. These patients demonstrate that TCR-engineered T cells can have serious and not readily predictable off-target and organ-specific toxicities and highlight the need for improved methods to define the specificity of engineered TCRs.

Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia

CAR T cells persist and sustain remissions in advanced chronic lymphocytic leukemia. CAR T cells for the long haul Immunotherapy is one of the most promising avenues of cancer therapy, with the potential to induce sustained remissions in patients with refractory disease. Studies with chimeric antigen receptor (CAR)–modified T cells have paved the way in patients with relapsed and refractory chronic lymphocytic leukemia. Porter et al. now report the mature results from their initial CAR T cell trial. CAR T cell persistence correlated with clinical responses, and these cells were functional up to 4 years after treatment. No patient who achieved complete remission relapsed, and no minimal residual disease was detected, suggesting that in a subset of patients, CAR T cells may drive disease eradication. Patients with multiply relapsed or refractory chronic lymphocytic leukemia (CLL) have a poor prognosis. Chimeric antigen receptor (CAR)–modified T cells targeting CD19 have the potential to improve on the low complete response rates with conventional therapies by inducing sustained remissions in patients with refractory B cell malignancies. We previously reported preliminary results on three patients with refractory CLL. We report the mature results from our initial trial using CAR-modified T cells to treat 14 patients with relapsed and refractory CLL. Autologous T cells transduced with a CD19-directed CAR (CTL019) lentiviral vector were infused into patients with relapsed/refractory CLL at doses of 0.14 × 108 to 11 × 108 CTL019 cells (median, 1.6 × 108 cells). Patients were monitored for toxicity, response, expansion, and persistence of circulating CTL019 T cells. The overall response rate in these heavily pretreated CLL patients was 8 of 14 (57%), with 4 complete remissions (CR) and 4 partial remissions (PR). The in vivo expansion of the CAR T cells correlated with clinical responses, and the CAR T cells persisted and remained functional beyond 4 years in the first two patients achieving CR. No patient in CR has relapsed. All responding patients developed B cell aplasia and experienced cytokine release syndrome, coincident with T cell proliferation. Minimal residual disease was not detectable in patients who achieved CR, suggesting that disease eradication may be possible in some patients with advanced CLL.

Chimeric Antigen Receptor T Cells against CD19 for Multiple Myeloma

A patient with refractory multiple myeloma received an infusion of CTL019 cells, a cellular therapy consisting of autologous T cells transduced with an anti-CD19 chimeric antigen receptor, after myeloablative chemotherapy (melphalan, 140 mg per square meter of body-surface area) and autologous stem-cell transplantation. Four years earlier, autologous transplantation with a higher melphalan dose (200 mg per square meter) had induced only a partial, transient response. Autologous transplantation followed by treatment with CTL019 cells led to a complete response with no evidence of progression and no measurable serum or urine monoclonal protein at the most recent evaluation, 12 months after treatment. This response was achieved despite the absence of CD19 expression in 99.95% of the patients neoplastic plasma cells. (Funded by Novartis and others; ClinicalTrials.gov number, NCT02135406.).

Bone marrow fibrosis: pathophysiology and clinical significance of increased bone marrow stromal fibres

In bone marrow biopsies, stromal structural fibres are detected by reticulin and trichrome stains, routine stains performed on bone marrow biopsy specimens in diagnostic laboratories. Increased reticulin staining (reticulin fibrosis) is associated with many benign and malignant conditions while increased trichrome staining (collagen fibrosis) is particularly prominent in late stages of severe myeloproliferative diseases or following tumour metastasis to the bone marrow. Recent evidence has shown that the amount of bone marrow reticulin staining often exhibits no correlation to disease severity, while the presence of type 1 collagen, as detected by trichrome staining, is often associated with more severe disease and a poorer prognosis. It was originally thought that increases in bone marrow stromal fibres themselves contributed to the haematopoietic abnormalities seen in certain diseases, but recent studies suggest that these increases are a result of underlying cellular abnormalities rather than a cause. A growing body of evidence suggests that increased deposition of bone marrow stromal fibres is mediated by transforming growth factor‐β and other factors elaborated by megakaryocytes, but it is likely that other cells, cytokines and growth factors are also involved. This suggests new avenues for investigation into the pathogenesis of various disorders associated with increased bone marrow stromal fibres.

Myeloproliferative Disorders and Myelodysplastic Syndromes

The myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS) encompass a pathologically and clinically heterogeneous group of hematologic entities that are united by their putative origin from pluripotent hematopoietic stem cells. The World Health Organization classifies these entities into three broad categories: (1) the chronic myeloproliferative diseases, (2) the myelodysplastic syndromes, and (3) the myelodysplastic/myeloproliferative diseases1 (see Table 35-1). Though all are true hematopoietic stem cell disorders characteristically associated with bone marrow hyperplasia, they are divergent in that MPD typically are associated with effective hematopoiesis, while MDS are associated with ineffective hematopoiesis, reflected by high or low peripheral blood counts, respectively.

A robust xenotransplantation model for acute myeloid leukemia.

Xenotransplantation of human acute myeloid leukemia (AML) in immunocompromised animals has been critical for defining leukemic stem cells. However, existing immunodeficient strains of mice have short life spans and low levels of AML cell engraftment, hindering long-term evaluation of primary human AML biology. A recent study suggested that NOD/LtSz-scid IL2Rγc null (NSG) mice have enhanced AML cell engraftment, but this relied on technically challenging neonatal injections. Here, we performed extensive analysis of AML engraftment in adult NSG mice using tail vein injection. Of the 35 AML samples analyzed, 66% showed bone marrow engraftment over 0.1%. Further, 37% showed high levels of engraftment (>10%), with some as high as 95%. A 2–44-fold expansion of AML cells was often seen. Secondary and tertiary recipients showed consistent engraftment, with most showing further AML cell expansion. Engraftment did not correlate with French–American–British subtype or cytogenetic abnormalities. However, samples with FLT3 mutations showed a higher probability of engraftment than FLT3 wild type. Importantly, animals developed organomegaly and a wasting illness consistent with advanced leukemia. We conclude that the NSG xenotransplantation model is a robust model for human AML cell engraftment, which will allow better characterization of AML biology and testing of new therapies.

Polymerase Chain Reaction Versus Southern Blot Hybridization Detection of Immunoglobulin Heavy-Chain Gene Rearrangements

To determine efficiently the clonality of B-cell lymphoproliferative disorders, we modified an immunoglobulin heavy-chain (IGH) gene rearrangement polymerase chain reaction (PCR) assay that requires only a single primer germline variable (VH) and joining (JH) pair and does not involve nested priming, blot hybridization, radioactivity, or sequencing of the amplified PCR product. This simple PCR technique enabled detection of IGH gene rearrangements in as little as 10 pg (one cell equivalent) of DNA or when the clonal-to-polyclonal B-cell ratio was experimentally set at 1:1000. We detected IGH gene rearrangements in 83.5% (71 of 85) of clonal B-cell processes, a sensitivity approaching that of more cumbersome multiple primer and nested primer assays. Moreover, this technique is equally effective with fixed tissues, either B5 or formalin, and can be performed on minute samples, histologic sections, fine-needle aspirates, or cerebrospinal fluids. When compared with conventional Southern blot analysis using a genomic JH probe, the PCR assay demonstrated IGH gene rearrangements in 82% (37 of 45) of B-cell processes positive by Southern blot. No false-positive results were observed in 29 negative control tissues. We now use IGH gene PCR routinely in our laboratory for the detection of clonal B-cells in virtually any tissue sample as an aid in early diagnosis, staging, and monitoring, and the Southern blot procedure is reserved for only a minority of diagnostic cases. for only a minority of diagnostic cases.

Immunoglobulin Heavy Chain Gene Analysis in Lymphomas: A Multi-Center Study Demonstrating the Heterogeneity of Performance of Polymerase Chain Reaction Assays

Determination of monoclonality through an evaluation of immunoglobulin heavy chain (IgH) gene rearrangements is a commonly performed and useful diagnostic assay. Many laboratories that perform this assay do so by the polymerase chain reaction (PCR). To evaluate current methods for performing IgH gene testing, 19 different Association of Molecular Pathology (AMP) member laboratories analyzed 29 blinded B cell and T cell lymphoid neoplasm samples of extracted DNA and formalin-fixed, paraffin-embedded (FFPE) tissue and were asked to complete a technical questionnaire. From this study, it is clear that Southern blot analysis remains the diagnostic gold standard, with a 100% diagnostic sensitivity and specificity. There was, however, remarkable heterogeneity in the performance of, and results obtained from, IgH PCR assays with diagnostic sensitivity ranging from over 90% to as low as 20%, when evaluating the same specimens. Many laboratories overestimate the diagnostic sensitivity of their IgH PCR assay, and there was a significant, and under appreciated, drop-off (from 61.3% to 41.8%) in detection in paired FFPE as compared with fresh/frozen tissues. Fixation has a dramatic impact on the inability to perform the test on FFPE (43.1%) versus DNA already extracted from fresh or frozen tissue (2.8%). A number of variables that affected the outcome of IgH PCR were identified. Strategies that improved the detection of monoclonal IgH rearrangements include: the addition of FRII to the FRIII upstream primer (increasing detection from 57.3% to 73.6%) and the use of the FR3A rather than the FR3 FRIII primer (increasing detection from 54.7% to 69.7%). Although numerous variables (from DNA extraction to PCR product detection) were evaluated, making it difficult to mandate alterations in laboratory practice, these findings ought to prompt diagnostic molecular pathology laboratories to reevaluate their claims of sensitivity, as well as their methodologies. Both pathologists and surgeons need to ensure that not all submitted material is fixed, if there is adequate sample. Importantly, there is a need for greater standardization to reduce the unacceptably high false negative rate of this crucial diagnostic assay.