RNA Loading of Leukemic Antigens into Cord Blood–Derived Dendritic Cells for Immunotherapy

The manipulation of dendritic cells (DCs) ex vivo to present tumor-associated antigens for the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTLs) attempts to exploit these cells’ pivotal role in immunity. However, significant improvements are needed if this approach is to have wider clinical application. We optimized a gene delivery protocol via electroporation for cord blood (CB) CD34+ DCs using in vitro–transcribed (IVT) mRNA. We achieved > 90% transfection of DCs with IVT-enhanced green fluorescent protein mRNA with > 90% viability. Electroporation of IVT-mRNA up-regulated DC costimulatory molecules. DC processing and presentation of mRNA-encoded proteins, as major histocompatibility complex/peptide complexes, was established by CTL assays using transfected DCs as targets. Along with this, we also generated specific antileukemic CTLs using DCs electroporated with total RNA from the Nalm-6 leukemic cell line and an acute lymphocytic leukemia xenograft. This significant improvement in DC transfection represents an important step forward in the development of immunotherapy protocols for the treatment of malignancy.

Brain tumour stem cells

The dogma that the genesis of new cells is a negligible event in the adult mammalian brain has long influenced our perception and understanding of the origin and development of CNS tumours. The discovery that new neurons and glia are produced throughout life from neural stem cells provides new possibilities for the candidate cells of origin of CNS neoplasias. The emerging hypothesis is that alterations in the cellular and genetic mechanisms that control adult neurogenesis might contribute to brain tumorigenesis, thereby allowing the identification of new therapeutic strategies.

Polarized secretion of leukemia inhibitory factor

Background: The direction of cytokine secretion from polarized cells determines the cytokine's cellular targets. Leukemia inhibitory factor LIF) belongs to the interleukin-6 IL-6) family of cytokines and signals through LIFR/gp130. Three factors which may regulate the direction of LIF secretion were studied: the site of stimulation, signal peptides, and expression levels. Stimulation with IL-1 beta is known to promote IL-6 secretion from the stimulated membrane apical or basolateral) in the human intestinal epithelial cell line Caco-2. Since LIF is related to IL-6, LIF secretion was also tested in Caco-2 following IL-1 beta stimulation. Signal peptides may influence the trafficking of LIF. Two isoforms of murine LIF, LIF-M and LIF-D, encode different signal peptides which have been associated with different locations of the mature protein in fibroblasts. To determine the effect of the signal peptides on LIF secretion, secretion levels were compared in Madin-Darby canine kidney MDCK) clones which expressed murine LIF-M or LIF-D or human LIF under the control of an inducible promoter. Low and high levels of LIF expression were also compared since saturation of the apical or basolateral route would reveal specific transporters for LIF. Results: When Caco-2 was grown on permeable supports, LIF was secreted constitutively with around 40% secreted into the apical chamber. Stimulation with IL-1 beta increased LIF production. After treating the apical surface with IL-1 beta, the percentage secreted apically remained similar to the untreated, whereas, when the cells were stimulated at the basolateral surface only 20% was secreted apically. In MDCK cells, an endogenous LIF-like protein was detected entirely in the apical compartment. The two mLIF isoforms showed no difference in their secretion patterns in MDCK. Interestingly, about 70% of murine and human LIF was secreted apically from MDCK over a 400-fold range of expression levels within clones and a 200,000-fold range across clones. Conclusion: The site of stimulation affected the polarity of LIF secretion, while, signal peptides and expression levels did not. Exogenous LIF is transported in MDCK without readily saturated steps.

Multidimensional classification and diagnosis of leukemia in flow cytometry

This research is to establish new optimization methods for pattern recognition and classification of different white blood cells in actual patient data to enhance the process of diagnosis. Beckman-Coulter Corporation supplied flow cytometry data of numerous patients that are used as training sets to exploit the different physiological characteristics of the different samples provided. The methods of Support Vector Machines (SVM) and Artificial Neural Networks (ANN) were used as promising pattern classification techniques to identify different white blood cell samples and provide information to medical doctors in the form of diagnostic references for the specific disease states, leukemia. The obtained results prove that when a neural network classifier is well configured and trained with cross-validation, it can perform better than support vector classifiers alone for this type of data. Furthermore, a new unsupervised learning algorithm---Density based Adaptive Window Clustering algorithm (DAWC) was designed to process large volumes of data for finding location of high data cluster in real-time. It reduces the computational load to ∼O(N) number of computations, and thus making the algorithm more attractive and faster than current hierarchical algorithms.

Genetic markers, birth characteristics, and childhood leukemia risk

The cause for childhood acute lymphoblastic leukemia (ALL) remains unknown, but male gender is a risk factor, and among ethnicities, Hispanics have the highest risk. In this dissertation, we explored correlations among genetic polymorphisms, birth characteristics, and the risk of childhood ALL in a multi-ethnic sample in 161 cases and 231 controls recruited contemporaneously (2007-2012) in Houston, TX. We first examined three lymphoma risk markers, since lymphoma and ALL both stem from lymphoid cells. Of these, rs2395185 showed a risk association in non-Hispanic White males (OR=2.8, P=0.02; P interaction=0.03 for gender), but not in Hispanics. We verified previously known risk associations to validate the case-control sample. Mutations of HFE (C282Y, H63D) were genotyped to test whether iron-regulatory gene (IRG) variants known to elevate iron levels increase childhood ALL risk. Being positive for either polymorphism yielded only a modestly elevated OR in males, which increased to 2.96 (P=0.01) in the presence of a particular transferrin receptor (TFRC) genotype for rs3817672 (Pinteraction=0.04). SNP rs3817672 itself showed an ethnicity-specific association (P interaction=0.02 for ethnicity). We then examined additional IRG SNPs (rs422982, rs855791, rs733655), which showed risk associations in males (ORs=1.52 to 2.60). A polygenic model based on the number of polymorphic alleles in five IRG SNPs revealed a linear increase in risk (OR=2.00 per incremental change; P=0.002). Having three or more alleles compared with none was associated with increased risk in males (OR=4.12; P=0.004). Significant risk associations with childhood ALL was found with birth length (OR=1.18 per inch, P=0.04), high birth weight (>4,000g) (OR=1.93, P=0.01), and with gestational age (OR=1.10 per week, P=0.04). We observed a negative correlation between HFE SNP rs9366637 and gestational age (P=0.005), again, stronger in males ( P=0.001) and interacting with TFRC (P interaction=0.05). Our results showed that (i) ALL risk markers do not show universal associations across ethnicities or between genders, (ii) IRG SNPs modify ALL risk presumably by their effects on iron levels, (iii) a negative correlation between an HFE SNP and gestational age exists, which implicates an iron-related mechanism. The results suggest that currently unregulated supplemental iron intake may have implications on childhood ALL development.

Prevention of oral lesions in children with acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children and is responsible for severe stomatologic complications. Treatment consists of four phases of chemotherapy, the main side effect of methotrexate, the drug most used during the intensification phase, is oral mucositis. OBJECTIVE: To evaluate the clinical aspects of the oral mucosa of children with ALL and to determine the effect of 0.12% chlorhexidine gluconate on the prevention of stomatologic complications in these patients. PATIENTS AND METHODS: Thirty-three children treated for ALL ranging in age from 2 to 15 years, without distinction of gender or race, were submitted to visual examination, digital palpation of the oral mucosa and cytologic examination of the buccal mucosa, and divided into two groups: group I consisted of 23 children using an oral solution of 0.12% chlorhexidine gluconate twice a day, and group II consisted of 10 children who did not receive this solution. All children received daily oral hygiene care guided by the dentist throughout treatment. RESULTS: Mucositis was observed in six children of group I and eight of group II, and was characterized by erythema, edema and ulcers. Uniform cytologic findings were obtained for the two groups, with a clear predominance of cells of the intermediate layer in all smears, in addition to a perinuclear halo in 18% of the smears. CONCLUSION: The present results suggest that systematic preventive treatment with 0.12% chlorhexidine gluconate and oral hygiene care reduce the occurrence of oral complications in children with ALL undergoing antineoplastic chemotherapy.

Prevention of oral lesions in children with acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children and is responsible for severe stomatologic complications. Treatment consists of four phases of chemotherapy, the main side effect of methotrexate, the drug most used during the intensification phase, is oral mucositis. OBJECTIVE: To evaluate the clinical aspects of the oral mucosa of children with ALL and to determine the effect of 0.12% chlorhexidine gluconate on the prevention of stomatologic complications in these patients. PATIENTS AND METHODS: Thirty-three children treated for ALL ranging in age from 2 to 15 years, without distinction of gender or race, were submitted to visual examination, digital palpation of the oral mucosa and cytologic examination of the buccal mucosa, and divided into two groups: group I consisted of 23 children using an oral solution of 0.12% chlorhexidine gluconate twice a day, and group II consisted of 10 children who did not receive this solution. All children received daily oral hygiene care guided by the dentist throughout treatment. RESULTS: Mucositis was observed in six children of group I and eight of group II, and was characterized by erythema, edema and ulcers. Uniform cytologic findings were obtained for the two groups, with a clear predominance of cells of the intermediate layer in all smears, in addition to a perinuclear halo in 18% of the smears. CONCLUSION: The present results suggest that systematic preventive treatment with 0.12% chlorhexidine gluconate and oral hygiene care reduce the occurrence of oral complications in children with ALL undergoing antineoplastic chemotherapy.

Tyrosine kinase inhibitor insensitivity of non-cycling CD34+ human acute myeloid leukaemia cells with FMS-like tyrosine kinase 3 mutations.

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events. (Blood. 2009; 113: 646-648)

Characterising the TP53-deleted subgroup of chronic lymphocytic leukemia: an analysis of additional cytogenetic abnormalities detected by interphase fluorescence in situ hybridisation and array-based comparative genomic hybridisation.

Deletion of the TP53 gene on chromosome 17p13.1 is the prognostic factor associated with the shortest survival in CLL. We used array-based comparative genomic hybridisation (arrayCGH) to identify additional DNA copy number changes in peripheral blood samples from 74 LRF CLL4 trial patients, 37 with >or=5% and 37 without TP53-deleted cells. ArrayCGH reliably detected deletions on 17p, including the TP53 locus, in cases with >or=50%TP53-deleted cells detected by fluorescence in situ hybridisation, plus seven additional cases with deleted regions on 17p excluding TP53. Losses on chromosomal regions 18p and/or 20p were found exclusively in cases with >or=5%TP53-deleted cells (por=5%TP53-deleted cases (p=0.02). In particular, amplification of 2p and deletion of 6q were both more frequent. Cases with >20%TP53-deleted cells had the worst prognosis in the LRF CLL4 trial.

Molecular characterization of complete and incomplete immunoglobulin heavy chain gene rearrangements in hairy cell leukemia.

PURPOSE: We analyzed patients with hairy cell leukemia (HCL) to achieve a better understanding of the differentiation stage reached by HCL cells and to define the key role of the diversification of cell surface makers, especially CD25 expression. PATIENTS AND METHODS: We analyzed 38 previously untreated patients with HCL to characterize their complete (VDJ(H)) and incomplete (DJ(H)) immunoglobulin (Ig) heavy chain (IgH) rearrangements, including somatic hypermutation pattern and gene segment use. RESULTS: A correlation between immunophenotypic profile and molecular data was seen. All 38 cases showed monoclonal amplifications: VDJ(H) in 97%, DJ(H) in 42%, and both in 39%. Segments from the D(H)3 family were used more in complete compared with incomplete rearrangements (45% vs. 12%; P <.005). Furthermore, comparison between molecular and immunophenotypic characteristics disclosed differences in the expression of CD25 antigen; CD25(-) cases, a phenotype associated with HCL variant, showed complete homology to the germline in 3 of 5 cases (60%), whereas this characteristic was never observed in CD25(+) cases (P <.005). Moreover, V(H)4-34, V(H)1-08, and J(H)3 segments appeared in 2, 1, and 2 CD25(-) cases, respectively, whereas they were absent in all CD25(+) cases. CONCLUSION: These results support that HCL is a heterogeneous entity including subgroups with different molecular characteristics, which reinforces the need for additional studies with a larger number of patients to clarify the real role of gene rearrangements in HCL.

The detection of contaminating clonal cells in apheresis products is related to response and outcome in multiple myeloma undergoing autologous peripheral blood stem cell transplantation.

In the present paper, we report on the use of the heteroduplex PCR technique to detect the presence of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) in the apheresis products of patients with multiple myeloma (MM) undergoing autologous peripheral blood stem cell (APBSC) transplantation. Twenty-three out of 31 MM patients undergoing APBSC transplantation with VDJH segments clonally rearranged detected at diagnosis were included in the study. Samples of the apheresis products were PCR amplified using JH and VH (FRIII and FRII) consensus primers and subsequently analyzed with the heteroduplex technique, and compared with those obtained at diagnosis. 52% of cases yielded positive results (presence of clonally rearranged VDJH segments in at least one apheresis). The presence of positive results in the apheresis products was not related to any pretransplant characteristics with the exception of response status at transplant. Thus, while no one patient with positive apheresis products was in complete remission (CR), negative immunofixation, before the transplant, five cases (46%) with negative apheresis were already in CR at transplant (P = 0.01). The remaining six cases with heteroduplex PCR negative apheresis were in partial remission before transplant. Patients with clonally free products were more likely to obtain CR following transplant (64% vs 17%, P= 0.02) and a longer progression-free survival, (40 months in patients transplanted with polyclonal products vs 20 with monoclonal ones, P = 0.03). These results were consistent when the overall survival was considered, since it was better in those patients with negative apheresis than it was in those with positive (83% vs 36% at 5 years from diagnosis, P= 0.01). These findings indicate that the presence of clonality rearranged VDJH segments is related to the response and outcome in MM transplanted patients.

Synthetic lethal targeting of oncogenic transcription factors in acute leukemia by PARP inhibitors

Acute myeloid leukemia (AML) is mostly driven by oncogenic transcription factors, which have been classically viewed as intractable targets using small molecule inhibitor approaches. Here, we demonstrate that AML driven by repressive transcription factors including AML1-ETO and PML-RARα are extremely sensitive to Poly (ADP-ribose) Polymerase (PARP) inhibitor (PARPi), in part due to their suppressed expression of key homologous recombination genes and thus compromised DNA damage response (DDR). In contrast, leukemia driven by MLL fusions with dominant transactivation ability is proficient in DDR and insensitive to PARP inhibition. Intriguing, depletion of an MLL downstream target, Hoxa9 that activates expression of various HR genes, impairs DDR and sensitizes MLL leukemia to PARPi. Conversely, Hoxa9 over-expression confers PARPi resistance to AML1-ETO and PML-RARα transformed cells. Together, these studies describe a potential utility of PARPi-induced synthetic lethality for leukemia treatment and reveal a novel molecular mechanism governing PARPi sensitivity in AML.

Emergency emicolectomy for intestinal primary Aspergillosis in acute myeloid leukemia

ntestinal aspergillosis is an infection with a very high death rate especially in leukemic patients. Here we describe a case of a 46 years old woman with acute myeloid leukemia (LAM M5) who developed intestinal primary aspergillosis. This patient was diagnosed with LAM M5 through bone marrow aspiration and bone biopsy in March 2004. Symptoms of the disease were slight persistent fever, weight loss, asthenia, anemia, thrombocytopenia,and leukocytosis with high number of blasts in peripheral blood. After induction chemotherapy with ICE (Ifosfamide, Carboplatin, Etoposide), she developed neutropenia and high fever without apparent infective foci. She was treated with empiric antibiotic therapy, nevertheless she developed an intense diarrhea and ileo-cecal distention. Diagnostic exams didn’t show signs of a focal lesion. Despite the change in antibiotic treatment and the transfusions of granulocytes and blood cells, the patient developed extremely critical conditions with persistence of neutropenia and abdominal distention. A surgical treatment was decided at the time. We treated the patient with a two steps surgical procedure. The first step was a right abdominal ileostomy followed by improvement of general conditions and then the second step a right colectomy. The histological morphology confirmed necrotizing colitis with Aspergillus ife. At that time , treatment with voriconazole was started. The general conditions of the patient improved rapidly and we were able to treat the patient with other medical anti-leukemic therapies. The patient is now cured and in healthy state. We obtained a good clinical result as only in other few cases described in literature.