Normalization of clonal diversity in gene therapy studies using shape constrained splines.

Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system. Hematopoietic cells originating from genetically modified stem cells will harbor insertions in specific genomic positions called integration sites, which represent unique genetic marks of clonal identity. Therefore, the analysis of vector integration sites present in the genomic DNA of circulating cells allows to determine the number of clones in the blood ecosystem. Shannon diversity index is adopted to evaluate the heterogeneity of the transduced population of gene corrected cells. However, this measure can be affected by several technical variables such as the DNA amount used and the sequencing depth of the library analyzed and therefore the comparison across samples may be affected by these confounding factors. We developed an advanced spline-regression approach that leverages on confounding effects to provide a normalized entropy index. Our proposed method was first validated and compared with two state of the art approaches in a specifically designed in vitro assay. Subsequently our approach allowed to observe the expected impact of vector genotoxicity on entropy level decay in an in vivo model of hematopoietic stem cell gene therapy based on tumor prone mice.

Shedding of clinical-grade lentiviral vectors is not detected in a gene therapy setting.

Gene therapy using viral vectors that stably integrate into ex vivo cultured cells holds great promises for the treatment of monogenic diseases as well as cancer. However, carry-over of infectious vector particles has been described to occur upon ex vivo transduction of target cells. This, in turn, may lead to inadvertent spreading of viral particles to off-target cells in vivo, raising concerns for potential adverse effects, such as toxicity of ectopic transgene expression, immunogenicity from in vivo transduced antigen-presenting cells and, possibly, gene transfer to germline cells. Here, we have investigated factors influencing the extent of lentiviral vector (LV) shedding upon ex vivo transduction of human hematopoietic stem and progenitor cells. Our results indicate that, although vector carry-over is detectable when using laboratory-grade vector stocks, the use of clinical-grade vector stocks strongly decreases the extent of inadvertent transduction of secondary targets, likely because of the higher degree of purification. These data provide supportive evidence for the safe use of the LV platform in clinical settings.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia.

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.

Vitamin A deficiency alters the bioelectric parameters and RNA content of rat gastric mucosa in vitro.

The present study was aimed to investigate the mechanisms by which vitamin A plays a role in maintaining the efficiency of gastric mucosal barrier. Particularly, we measured electrical parameters and the RNA/DNA ratio of gastric mucosa isolated in vitro from the stomach of rats in which vitamin A-deficiency was induced by means of a vitamin A-free diet and then abolished by means of a massive vitamin A supplementation. Pair-fed vitamin A-nondepleted rats and normal rats fed ad libitum on a standard diet served as controls. Vitamin A status was assayed for each group of rats by measuring the hepatic content of vitamin A. We found that in gastric mucosa vitamin A-deficiency induced: 1) a decrease in both transmucosal potential difference and short-circuit current; 2) an increase in transmucosal electrical resistance; 3) a decrease in RNA content resulting in a decreased RNA/DNA ratio. Abolishment of vitamin A-deficiency restored both electrical parameters and RNA content of rat gastric mucosa. Our results stress the role of vitamin A in maintaining the efficiency of the gastric mucosal barrier. Vitamin A seems to act by stabilizing gastric electrical parameters and by controlling the protein synthesis/turnover in the surface gastric mucosal cells.

Ex vivo priming for long-term maintenance of antileukemia human cytotoxic T cells suggests a general procedure for adoptive immunotherapy.

Adoptive cellular immunotherapy has proven to be a successful approach in preventing and curing cytomegalovirus infection and Epstein-Barr virus-associated lymphomas after bone marrow transplantation. Translation of this approach for preventing leukemia relapse after bone marrow transplantation might require ex vivo priming and long-term maintenance of leukemia blast-specific T cells. To accomplish this goal, procedures were optimized for the in vitro priming of naive CD8 using dendritic cells activated by CD40 ligation, interleukin-12 (IL-12), and IL-7. Using T lymphocytes and dendritic cells obtained from HLA-matched allogeneic bone marrow transplantation donors and leukemia blasts as a source of tumor antigens, anti-acute myeloid leukemia cytotoxic T lymphocytes (CTLs) were induced. In these experiments, it was found that though it is possible to induce CTLs using immature dendritic cells, IL-12, and IL-7, obtaining long-term CTLs requires the presence of CD4 T cells in the priming phase. Using this approach, long-term antileukemia CTL lines could be generated from 4 of 4 bone marrow donors. Because this procedure does not require definition of the target antigen and because it selects responding cells from a virgin T-cell repertoire, its general application is suggested in adoptive immunotherapy and in the definition of tumor rejection antigens.

Solitary breast metastases from a renal cell carcinoma.

A case of solitary and metachronous breast metastases from a renal cell carcinoma is described nine years after surgery. The review of the literature proves that the breast is an unusual site for metastatic disease.

Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6.

Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression.

MID2, a homologue of the Opitz syndrome gene MID1: similarities in subcellular localization and differences in expression during development.

The B-box family is an expanding new family of genes encoding proteins involved in diverse cellular functions such as developmental patterning and oncogenesis. A member of this protein family, MID1, is the gene responsible for the X-linked form of Opitz G/BBB syndrome, a developmental disorder characterized by defects of the midline structures. We now report the identification of MID2, a new transcript closely related to MID1. MID2 maps to Xq22 in human and to the syntenic region on the mouse X chromosome. The two X-linked genes share the same domains, the same exon-intron organization, a high degree of similarity at the protein level and the same subcellular localization, both being confined to the cytoplasm in association to micro-tubular structures. The expression pattern studied by RNA in situ hybridization in mouse revealed that Mid2 is expressed early in development and the highest level of expression is detected in the heart, unlike Mid1 for which no expression was detected in the developing heart. Together, these data suggest that midin and MID2 have a similar biochemical function but a different physiological role during development.

Characterisation of CTL directed towards non-inherited maternal alloantigens in human cord blood.

Allogeneic cord blood transplantation (CBT), especially from unrelated donors, is being increasingly used for treating paediatric patients with both malignant and non-malignant disorders. Recent clinical and experimental evidence suggests that human cord blood mononuclear cells (CBMC) may acquire in utero a state of tolerance towards non-inherited maternal antigens (NIMA). In order to better define this phenomenon, we measured, by means of a limiting dilution assay (LDA), the frequency of NIMA-specific CTL precursors (CTLp) in cord blood samples obtained from 13 healthy neonates. The immunophenotype of the effector cells recovered from LDA was also analysed. Data concerning both CTLp frequency and phenotype of effector cells were compared with those obtained stimulating CBMC with cells of paternal origin (NIPA) and adult PBMC with allogeneic targets. Results showed that cytotoxic cells directed towards cells of maternal origin could be detected in all cord blood samples tested. Phenotype analysis demonstrated that NIPA elicit the expansion of CD3+/CD8bright T cells, a phenotype associated with alloreactive CTL. By contrast, NIMA preferentially stimulated the expansion of CD3-/CD8dim+ cells, a phenotype associated with NK cells, which are known to be able, in certain clinical conditions, to kill allogeneic haematopoietic cells without causing GVHD. Thus, our results indicate that, when evaluated in a limiting dilution condition, NIMA-reactive cord blood cells are detectable and a preferential expansion of NK cells is observed.

Mitomycin-C and lonidamine as second-line therapy for colorectal cancer: a phase II study.

AIMS AND BACKGROUND: Recent preclinical data have suggested that lonidamine may potentiate the acitivity of mitomycin C in human colon cancer cell lines LoVo and HT29. STUDY DESIGN: A phase II study was carried out in 14 patients with advanced colorectal cancer pretreated with fluorouracil and folinic acid. Treatment consisted of lonidamine, 600 mg po, followed after 2 h by mitomycin, 20 mg/m2 by iv bolus, followed by lonidamine, 150 mg tio for 5 days; the cycle was repeated every 6 weeks. RESULTS: No objective response was seen. Three patients had stable disease; the median survival for the whole group was 4 months. Although hematologic toxicity was negligible, lonidamine-related side effects were moderate to severe in most patients and mainly represented by myalgia and gastric pain. DISCUSSION: Despite a sound preclinical rationale, this schedule of lonidamine and mitomycin C was ineffective and toxic in patients with advanced colorectal cancer. More experimental data about lonidamine are needed in order to design more effective regimens based on the combination of this interesting drug with other anticancer agents.

Identification of HLA-unrestricted CD8+/CD28- cytotoxic T-cell clones specific for leukemic blasts in children with acute leukemia.

We report on the identification of 57 T-cell clones (TCC) cytolytic to autologous leukemic blasts (LB) but not autologous bone marrow remission cells. LB-reactive TCC were obtained from 3 children with acute leukemia at remission; all expressed the same phenotype, CD3/TCR alpha beta/CD8+, but were heterogeneous for the expression of V beta T-cell receptor (TCR) V region chains, thus showing that these cells were not derived from the expansion of a single clone. Cytolytic activity of LB-reactive TCC was not restricted to autologous LB because they were also able to lyse phenotypically similar allogeneic LB but not bone marrow remission cells of the same patients. Neither autologous nor allogeneic LB used in the present study as stimulator and target cells expressed CD80 (B7/BB-1) antigen, and LB-reactive TCC were CD28-. Cytolytic activity of the clones was only inhibited by anti-CD11a (LFA-1) mAb but not by mAbs specific for HLA class I and II, CD3, CD8, or TCR alpha beta. In conclusion, these data suggest that a subset of apparently HLA-unrestricted, CD3/TCR alpha beta/CD8+ CD28- cytotoxic T lymphocytes, which use a TCR/CD3-independent recognition pathway, is primarily involved in antitumor immune response of children with acute leukemia at remission, possibly contributing to the control of minimal residual disease.

L-folinic acid and 5-fluorouracil in the treatment of advanced breast cancer: a phase II study.

BACKGROUND: The combination of 5-fluorouracil (5-FU) and folinic acid (FA) is an active combination for the treatment of advanced breast cancer (ABC). Theoretically, the biologically active isomer of FA, 1-FA, should be more effective than racemic FA in modulating 5-FU activity. PATIENTS AND METHODS: Thirty-three patients (pts) with ABC, all previously treated with an anthracycline-based combination for advanced disease were treated with 1-FA: 100 mg/m2 i.v. and 5-FU: 370 mg/m2 i.v. for 5 consecutive days every 4 weeks. RESULTS: Three complete remission (CR) and 11 partial remission (PR) were obtained for an overall response rate of 42% (95% CI = 25-59). Median duration of response was 10 months, median survival was 15 months for responders, 11 months for NC and 3 for PD. Eleven pts experienced a WHO grade III-IV oral mucositis (33%), 6 pts had grade III and one grade IV diarrhea, two pts had grade IV neutropenia resulting in one toxic death. CONCLUSIONS: In this heavily pretreated population of pts with ABC, this regimen showed an interesting activity with substantial toxicity. Both the response rate and the pattern of side-effects seem similar to those experienced with the racemic mixture of d,1-FA. Modulated 5-FU warrants an increasing consideration in the treatment of breast cancer.

Fluorouracil, high-dose folinic acid, low-dose alpha-2b interferon and dipyridamole in the treatment of advanced colorectal cancer. A pilot study.

Twenty-six patients with advanced colorectal cancer were treated with a combination based on multimodal biochemical modulation of 5-fluorouracil by means of high dose folinic acid, low-dose alpha-2b interferon and dipyridamole. The overall response rate was 42% (95% confidence intervals = 23%. 61%) with four complete remissions (15%). The median duration of response was 9 months and the median survival for responders was 15 months (all patients = 12 months). Toxic side effects included oral mucositis (grade III-IV = 38%) and a generally mild flu-like syndrome. This regimen seems active and safe enough to be compared with the combination of fluorouracil and high-dose folinic acid.

Mitomycin-C, vinblastine, and lonidamine as salvage treatment of advanced breast cancer. A pilot study.

Thirty-two patients with advanced breast cancer were treated with mitomycin-C 10 mg/m2 IV and vinblastine 6 mg/m2 IV every 21 days in combination with lonidamine 450 mg/day P.O. and prednisone 15 mg/day P.O. given continuously. Among the 29 evaluable patients (all but three pretreated with an anthracycline-based regimen), one complete remission (CR) and six partial remissions (PR) (response rate, 24%) were seen. The median duration of response was 14 months (range, 4-30 months). Median survival for responders was 18 months (range, 4-30 months). Hematological toxicity was uncommon; the main lonidamine-related side effects were myalgia, abdominal cramps, and reversible deafness; these side effects were severe in two, one, and one patients, respectively. The regimen seems to have a reasonable degree of activity and toxicity in advanced, refractory breast cancer. The role of lonidamine in the treatment of this disease warrants further evaluation.

Phase II trial of 5-fluorouracil and high-dose folinic acid in advanced renal cell cancer.

Fourteen patients with metastatic renal cell carcinoma (RCC) were treated with high-dose folinic acid (HDFA): 200 mg/m2 i.v. and 5-fluorouracil (5-FU): 370 mg/m2 i.v. for 5 consecutive days every 28 days. Severe oral mucositis (WHO grade III-IV) was experienced by two patients, whereas hematological toxicity was mild. No complete or partial remission was observed. Short-lasting stable disease occurred in 8 patients (median = 5 months, range 2-11). This combination does not need further evaluation in patients with RCC.

5-Fluorouracil and dipyridamole in metastatic breast cancer: a pilot study.

In an attempt to increase the clinical activity of 5-fluorouracil (5-FU) by blocking the thymidine salvage pathway, 15 patients with refractory metastatic breast cancer (MBC) were treated with oral dipyridamole (D): 75 mg p.o. q.i.d. and 5-FU: 400 mg/m2 i.v. by bolus for 5 consecutive days, every 28 days. All the patients were pretreated with 5-FU and an anthracycline-based regimen. Toxicity was minimal, with 8 patients experiencing a D-related moderate headache. Although no objective responses were seen, two patients with 5-FU refractory disease showed tumor shrinkage. A different D schedule and the addition of folinic acid (FA) to 5-FU might provide better results and deserves further evaluation.

Cyclophosphamide, epirubicin, high-dose folinic acid and 5-fluorouracil (super-FEC) as first-line chemotherapy for advanced breast cancer: preliminary results.

Forty patients with metastatic breast cancer were treated with a new combination regimen consisting of cyclophosphamide, epirubicin, high-dose folinic acid and 5-fluorouracil (super-FEC). A major objective response was observed in 32 patients (80%). Among these, 11 patients (27%) experienced a complete remission. The median duration of response was 10+ and 12+ months for CR and PR, respectively. The most common side-effect was oral mucositis (Grade III = nine patients; grade IV = two patients), while haematological toxicity was virtually absent. Considering the high-risk characteristics of the vast majority of the enrolled patients (75% had dominant visceral disease), these preliminary results suggest that super-FEC has a powerful activity in poor-prognosis metastatic breast cancer with an acceptable degree of toxicity.

Complete estrogen blockade with buserelin and aminoglutethimide for advanced breast cancer: a phase I-II study with long-term hormonal correlations.

We treated 21 patients with advanced breast cancer with buserelin, aminoglutethimide and cortisone acetate in an attempt to obtain a complete estrogen blockade both in premenopausal and postmenopausal patients. Ten patients (47%) obtained an objective response without any relevant side-effects. Dealing with hormonal data, it must be outlined that serum testosterone levels decreased significantly in postmenopausal patients, suggesting a possible explanation for the activity of luteinizing hormone-releasing hormone analogue in this group of patients.