Feasibility and safety of adoptive immunotherapy with ex vivo-generated autologous, cytotoxic T lymphocytes in patients with solid tumor.

BACKGROUND AIMS: Adoptive T-cell therapy with tumor-specific T cells has emerged as a potentially useful approach for treating patients with advanced malignancies. We have demonstrated previously the feasibility of obtaining large numbers of autologous anti-tumor-specific cytotoxic T lymphocytes (CTL) generated by stimulation of patients' peripheral blood mononuclear cells with dendritic cells pulsed with apoptotic tumor cells. Methods. Six patients with progressing metastatic solid tumors (one renal cell carcinoma, two ovarian cancers, two extraosseous peripheral neuroectodermal tumors, one soft tissue sarcoma) not eligible for conventional therapies were treated with adoptive immunotherapy. Anti-tumor CTL, proven to be reactive in vitro against patient tumor cells, but not against normal cells, were infused following lymphodepleting chemotherapy administered to favor T-cell proliferation in vivo. RESULTS: Patients received a median of nine CTL infusions (range 2-19). The median number of CTL administered per infusion was 11 × 10(8) (range 1-55 × 10(8)). No patient experienced acute or late adverse events related to CTL infusion, even when large numbers of cells were given. Post-infusion laboratory investigations demonstrated an increase in the frequency of circulating anti-tumor T-cells and, in patients with a longer follow-up receiving two CTL infusions/year, a stabilization of these values. CONCLUSIONS: Our study demonstrates that autologous ex vivo-generated anti-tumor CTL can be administered safely in patients with advanced solid tumors and can improve the immunologic reactivity of recipients against tumor. These preliminary results provide a rationale for evaluating the clinical efficacy of this immunotherapeutic approach in phase I/II studies.

Impact of inflammatory cytokine gene polymorphisms on developing acute graft-versus-host disease in children undergoing allogeneic hematopoietic stem cell transplantation.

Single nucleotide polymorphisms (SNPs) in gene encoding pro- and anti-inflammatory factors have been associated with the occurrence of aGvHD. We retrospectively tested a wide panel of 38 polymorphisms in 19 immunoregulatory genes, aiming to first establish, in a pediatric HSCT setting, which SNPs were significantly associated with the development of aGvHD. A significant association was found between aGvHD grades II-IV and SNPs of donor IL10-1082GG, and Fas-670CC + CT and recipient IL18-607 TT + TG genotype. aGvHD grades III-IV resulted associated with donor IL10-1082GG, Fas-670CC + CT, and TLR4-3612TT as well as the use of peripheral CD34+ cells as stem cell source. The multivariate analysis confirmed the association between donor IL10-1082GG and Fas-670CC + CT and aGvHD grades II-IV and between donor IL10-1082GG and TLR4-3612TT and aGvHD grades III-IV. In conclusion we found an association between IL10, FAS, and TLR4 in the donor and IL18 in the recipient and an increased risk of developing aGvHD in transplanted children. Knowledge of the SNPs of cytokine genes associated with aGvHD represents a useful tool for an integrated pretransplantation risk assessment and could guide the physicians to an optimal and more accurate HSCT planning.

Real-time RT-PCR of tyrosine hydroxylase to detect bone marrow involvement in advanced neuroblastoma.

Neuroblastoma (NB) is characterised by the secretion of catecholamines in approximately 95% of patients. Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis pathway. Expression of the tyrosine hydroxylase gene (TH) is regulated in a tissue-specific manner during neonatal development and differentiation, therefore TH mRNA expression is a specific tumour marker for NB. Here we present a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using TaqMan technology for detection and quantification of TH mRNA in bone marrow (BM) NB patients. The degree of TH expression was derived from the ratio of the mRNAs of this gene and the reference gene, beta-actin. A ratio greater than 3x10(-2) was considered as positive for TH mRNA presence. Samples were also examined for TH mRNA by first and nested RT-PCR. Seventeen BM samples from 4 patients with disseminated NB (3 stage IV and 1 stage IVs) were evaluated at diagnosis and during treatment. We found a variable degree of TH expression ranging from 0.0344 to 26.3370 in 12/17 positive samples, while no TH mRNA (value lower than 3x10(-2)) was detected in 5/17 samples obtained after consolidation therapy. Our results show a moderate concordance between different qualitative RT-PCR methods and real-time RT-PCR. The real-time RT-PCR results seem to fit better with the natural short-term clinical follow-up of the evaluated patients, with respect to qualitative methods. Real-time TH RT-PCR could therefore be of clinical value for the assessment of a patient's prognosis by monitoring minimal residual disease (MRD).

Transient abnormal myelopoiesis in a phenotypically normal newborn with polyclonal trisomy 21.

We report a rare case of transient abnormal myelopoiesis (TAM) in a phenotypically normal neonate. The presence of a palpable hepatomegaly prompted in-depth laboratory tests, which revealed the presence of severe hyperleukocytosis, with blast cells present in a peripheral blood smear. Although no signs of Down syndrome were present, we suspected TAM. Further analysis identified a mutation in GATA1 along with the unique finding of two different trisomic cell lines, detected upon karyotyping; one with trisomy 21 only, and one with trisomies 21 and 22, which was present in a subpopulation of peripheral blood cells. These genetic abnormalities disappeared by the age of 6 months. The presence of two different trisomic clones may be an evidence of the polyclonal nature of TAM in this patient.

MYCN is a novel oncogenic target in pediatric T-cell acute lymphoblastic leukemia.

MYCN is an oncogene frequently overexpressed in pediatric solid tumors whereas few evidences suggest his involvement in the pathogenesis of haematologic malignancies. Here we show that MYCN is overexpressed in a relevant proportion (40 to 50%) of adult and pediatric T-cell acute lymphoblastic leukemias (T-ALL). Focusing on pediatric T-ALL, MYCN-expressing samples were found almost exclusively in the TAL1-positive subgroup. Moreover, TAL1 knockdown in T-ALL cell lines resulted in a reduction of MYCN expression, and TAL1 directly binds to MYCN promoter region, suggesting that TAL1 pathway activation could sustain the up-regulation of MYCN. The role of MYCN in T-ALL was investigated by peptide nucleic acid (PNA-MYCN)-mediated transcriptional silencing of MYCN and by siRNAs. MYCN knockdown in T-ALL cell lines resulted in a reduction of cell viability, up to 50%, while no effect was elicited with a mismatch PNA. The inhibitory effect of PNA-MYCN on cell viability was due to a significant increase in apoptosis. PNA-MYCN treatment in pediatric T-ALL samples reduced cell viability of leukemic cells from patients with high MYCN expression, while no effect was obtained in MYCN-negative blast cells. These results showed that MYCN is frequently overexpressed in pediatric T-ALL and suggested his role as a candidate for molecularly-directed therapies.