The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity.

The primary aim of this clinical trial was to determine the feasibility of delivering first-generation CAR T cell therapy to patients with advanced, CEACAM5+ malignancy. Secondary aims were to assess clinical efficacy, immune effector function and optimal dose of CAR T cells. Three cohorts of patients received increasing doses of CEACAM5+-specific CAR T cells after fludarabine pre-conditioning plus systemic IL2 support post T cell infusion. Patients in cohort 4 received increased intensity pre-conditioning (cyclophosphamide and fludarabine), systemic IL2 support and CAR T cells. No objective clinical responses were observed. CAR T cell engraftment in patients within cohort 4 was significantly higher. However, engraftment was short-lived with a rapid decline of systemic CAR T cells within 14 days. Patients in cohort 4 had transient, acute respiratory toxicity which, in combination with lack of prolonged CAR T cell persistence, resulted in the premature closure of the trial. Elevated levels of systemic IFNγ and IL-6 implied that the CEACAM5-specific T cells had undergone immune activation in vivo but only in patients receiving high-intensity pre-conditioning. Expression of CEACAM5 on lung epithelium may have resulted in this transient toxicity. Raised levels of serum cytokines including IL-6 in these patients implicate cytokine release as one of several potential factors exacerbating the observed respiratory toxicity. Whilst improved CAR designs and T cell production methods could improve the systemic persistence and activity, methods to control CAR T 'on-target, off-tissue' toxicity are required to enable a clinical impact of this approach in solid malignancies.

Next-generation sequencing of the TET2 gene in 355 MDS and CMML patients reveals low-abundance mutant clones with early origins, but indicates no definite prognostic value.

Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using "next-generation" sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) ≥ 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chronic myelomonocytic leukemia patients (P < .001). Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. "Deeper" sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34(+) and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (> 50 years) was associated with a higher incidence of TET2 mutation (P = .02).

Novel TET2 mutations associated with UPD4q24 in myelodysplastic syndrome.

PURPOSE: Cryptic chromosomal aberrations, such as regions of uniparental disomy (UPD), have been shown to harbor homozygous mutations and are a common feature in myelodysplastic syndrome (MDS). We investigated the sequence integrity of 4q24 candidate tumor suppressor gene TET2 in MDS patients with UPD on chromosome 4. PATIENTS AND METHODS: The coding exons of TET2 were analyzed by 454 deep sequencing and Sanger sequencing in nine patients with UPD on 4q. Four patients had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) and UPD4q24, and five patients (refractory anemia with excess blasts-II, n = 1; 5q- syndrome, n = 1; RCMD-RS, n = 1; refractory anemia, n = 1; refractory cytopenia with multilineage dysplasia, n = 1) had no UPD4q24. RESULTS: Mutations on TET2 were identified in all four patients with UPD4q24. These were localized to exons 3, 6, and 9 and resulted in two premature stop codons, one frameshift mutation, and one cysteine to glycine amino acid change. Mutant clone size varied between 30% and 85%. One patient with UPD outside of q24 (UPD4q28.3) displayed additional TET2 mutations, but these were at low clonal levels (13%, 4%, and 4% for a silent mutation, a 180-base pair deletion in exon 3, and a lysine to phenylalanine substitution in exon 11, respectively). The other patients who did not have UPD4q24 did not have verifiable TET2 mutations. CONCLUSION: Our data identify novel TET2 mutations in a dominant clone in patients with UPD4q24. The presence of UPD4q24 and mutations in RCMD-RS patients may suggest specificity to this subtype. Our preliminary results need to be confirmed in a large cohort of all MDS subtypes.

Anti-tumor immunity in a model of acute myeloid leukemia.

Whole-cell vaccines allow the induction of anti-tumor immune responses without the need to define tumor antigens. We wished to directly compare, for the first time, the capacity of B7-1, B7-2 and 4-1BB ligand (4-1BBL) costimulatory molecules to convert murine and human acute myeloid leukemia (AML) cells into whole vaccines. 32Dc-kit is a murine myeloid cell line, which develops an AML-like disease over a protracted period, emulating human AML disease development. 32Dc-kit cells were modified to express elevated levels of B7-1, B7-2 or 4-1BBL, and each led to tumor rejection, although only mice injected with 32Dc-kit/B7-2 cells were able to reject subsequent parental tumor cell challenge. T-cell deficient nude mice were able to reject the 32Dc-kit variants, but they could not reject parental cell challenge; however, we found no evidence of cytotoxic T lymphocyte or natural killer (NK) activity ex vivo suggesting that tumor cell killing was mediated by an immune response that could not be recapitulated using purified NK or T cells as lone effectors. In human allogeneic mixed lymphocyte reactions (MLRs), we found no single costimulatory molecule was more effective, suggesting that the induction of a universal anti-tumor response will require a combination of costimulatory molecules.

Prevalence of the activating JAK2 tyrosine kinase mutation V617F in the Budd-Chiari syndrome.

BACKGROUND & AIMS: Budd-Chiari Syndrome (BCS) results from obstruction to hepatic venous outflow, with myeloproliferative disorder (MPD) accounting for up to 40% of cases. A number of BCS cases labelled as "idiopathic" do not fulfill the diagnostic criteria for MPD but have features suggestive of a latent form based on hyperplastic bone marrow and erythroid progenitor cell culture; these cases may subsequently develop overt MPD. A clonal mutation in JAK2 tyrosine kinase (JAK2V617F) occurs in a high proportion of patients with MPD and is of use in the characterization of latent MPD in BCS. METHODS: We performed allele-specific polymerase chain reaction to screen for JAK2V617F in subjects with BCS (n = 41) and polycythemia vera (PV) (n = 20) and in hematologically normal controls (n = 27). RESULTS: AK2V617F was detected in 24 of 41 (58.5%) subjects with BCS, 19 of 20 PV controls, and 0 of 27 hematologically normal controls. Mean hemoglobin concentration and hematocrit were significantly higher in patients with JAK2V617F. Bone marrow was hyperplastic in 16 of 41 subjects (12/16 JAK2V617F positive). Nine of 33 (27.3%) showed endogenous erythroid colony formation (7/9 JAK2V617F positive). Eleven of 41 subjects developed overt MPD (8/11 essential thrombocythemia, 3/11 PV) after the diagnosis of BCS (median, 49 months; range, 8-87 months), and in 90.9% of these JAK2V617F was detected. CONCLUSIONS: JAK2V617F occurs in a high proportion of patients with BCS. Latent MPD was missed in a substantial number of our subjects by using standard techniques. Such cases should be screened for JAK2V617F and carefully observed for the subsequent development of overt MPD.

Microarray analysis of tumour antigen expression in presentation acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a difficult to treat disease, especially for those patients who have no eligible haematopoietic stem cell (HSC) donor. One of the most promising treatment options for these patients is immunotherapy. To investigate the expression of known tumour antigens in AML, we analysed microarray data from 124 presentation AML patient samples and investigated the present/absent calls of 82 tumour-specific or -associated antigens. We found 11 antigens which were expressed in AML patient samples but not normal donors. Nine of these were cancer-testis (CT) antigens, previously shown to be expressed in tumour cells and immunologically protected sites and at very low levels, if at all, in normal tissues. Expression was confirmed using real-time PCR. We have identified a number of CT antigens with expression in presentation AML samples but not normal donor samples, which may provide effective targets for future immunotherapy treatments early in disease.

Antiapoptotic microenvironment of acute myeloid leukemia.

We showed previously that tumor-derived supernatant (TSN) from acute myeloid leukemia (AML) myeloblasts inhibits peripheral blood T cell activation and proliferation, rendering the T cells functionally incompetent. We show here that the AML TSN also significantly delays apoptosis of both resting and stimulated T cells, as judged by reduction in annexin V/propidium iodide staining. In addition, we show that this is not unique to T cells and that AML TSN inhibits apoptosis of peripheral B cells, neutrophils, and monocytes. Furthermore, it also enhances the survival of other AML myeloblasts with lower viability. Investigations into the mechanism demonstrate a reduction in the cleavage of procaspase-3, -8, and -9 and the caspase substrate, poly(ADP-ribose)polymerase (PARP). This may be due to Bcl-2, which is normally down-regulated in CD3/CD28-stimulated T cells, but is maintained in the presence of AML TSN. We conclude that AML cells generate an antiapoptotic microenvironment that favors the survival of malignant cells, but also inhibits apoptosis of other normal hemopoietic cells. Reversal of these immunosuppressive effects and restoration of normal immune responses in patients with AML would improve the success of immunotherapy protocols.

Apoptosis in the myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are neoplastic dyscrasias characterized by peripheral cytopenia, despite a normocellular or hypercellular bone marrow. In the past decade, it has become apparent that this ineffective hemopoiesis is largely caused by excessive apoptosis of myeloid precursors. There is no evidence for gain-of-function mutations within the apoptotic machinery in MDS. It appears that the apoptosis is a reactive phenomenon fueled by cytokines. The provoking stimulus for the proapoptotic intramedullary milieu in MDS is unknown. The evolution of MDS from early relatively chronic to aggressive and frankly leukemic phenotype is accompanied by a suppression of apoptosis. This metamorphosis correlates with changes in intracellular levels of Bcl-2-family proteins, but the genetic basis for this shift has not been elucidated clearly. Expression profiling and proteomic technologies may offer the best means to unravel this process.

Serum erythropoietin values in erythrocytoses and in primary thrombocythaemia.

Serum erythropoietin (Epo) values were estimated in samples from 125 patients with erythrocytosis to examine the specificity and sensitivity of reduced and raised values in the diagnosis of polycythaemia vera (PV) and secondary erythrocytosis (SE) respectively. Additionally, Epo values were estimated in samples from 49 patients with primary thrombocythaemia (PT) to determine whether Epo values were altered. We found high specificity (92%) and moderate sensitivity (64%) of low serum Epo values (below the reference range) in the diagnosis of PV, and also poor sensitivity (47%) of raised Epo values in the diagnosis of SE. Raised Epo values were not observed in PV patients with Hb > 14.0 g/dl and were only observed in one PV patient with a relatively low Hb recovering from a gastro-intestinal haemorrhage. Raised Epo values occurred in some patients with apparent erythrocytosis (AE) and idiopathic erythrocytosis (IE), mainly at normal (rather than raised) Hb values (< 16 g/dl). Low Epo values occurred in a few AE, IE and SE patients at higher Hb values (> 16 g/dl). Low Epo values were less specific for PV when the Hb was raised, while raised Epo values were less specific for SE when the Hb was not raised. Approximately one third of patients with PT had a low (below the reference range) Epo value, this being associated with a high normal Hb (> 14 g/dl, P < 0.001) and showing a trend towards association with absence of treatment. The high normal Hb values were in turn associated with an increased incidence of thrombotic events (P < 0.05). These findings could influence the future investigation and management of PT patients.