Modeling tumor-host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, the B-cell receptor (BCR) and nuclear factor- κB (NF-κB) pathways are activated in the lymph node (LN) microenvironment. Thus, model systems mimicking tumor-host interactions are important tools to study CLL biology and pathogenesis. We investigated whether the recently established NOD/scid/γc(null) (NSG) mouse xenograft model can recapitulate the effects of the human microenvironment. We assessed, therefore, tumor characteristics previously defined in LN-resident CLL cells, including proliferation, and activation of the BCR and NF-κB pathways. We found that the murine spleen (SP) microenvironment supported CLL cell proliferation and activation to a similar degree than the human LN, including induction of BCR and NF-κB signaling in the xenografted cells. Next, we used this model to study ibrutinib, a Bruton's tyrosine kinase inhibitor in clinical development. Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment, decreased proliferation, induced apoptosis and reduced the tumor burden in vivo. Thus, our data demonstrate that the SP of xenografted NSG mice can, in part, recapitulate the role of the human LN for CLL cells. In addition, we show that ibrutinib effectively disrupts tumor-host interactions essential for CLL cell proliferation and survival in vivo.

Improved outcome following allogeneic stem cell transplantation in chronic myeloid leukemia is associated with higher expression of BMI-1 and immune responses to BMI-1 protein.

BMI-1 and EZH2 are polycomb group (PcG) proteins that maintain self-renewal of stem cells, and are overexpressed in leukemia. To investigate the potential of PcG proteins as leukemia-associated antigens, and as targets for graft-versus-leukemia (GVL) effects, we studied cells obtained from 86 patients with chronic myeloid leukemia (CML) and 25 human leukocyte antigen (HLA)-A*0201(+) sibling donors collected before allogeneic stem cell transplantation (SCT). Although BMI-1 overexpression in CD34(+) cells of CML patients treated with pharmacotherapy is associated with poor prognosis, we found, conversely, that in CML patients treated with SCT, a higher expression of BMI-1, and correspondingly a lower expression of its target for repression, CDKN2A, is associated with improved leukemia-free survival. Cytotoxic T-lymphocyte (CTL) responses to the BMI-1 peptide were detected in 5 of 25 (20%) donors, and in 8 of 19 (42%) HLA-A*0201(+) CML patients. BMI-1 generated more total and high-avidity immune responses, and was more immunogenic than EZH2. PcG-specific CTLs had a memory phenotype, were readily expanded in short-term cultures and were detected after SCT in recipients of PcG-specific CTL-positive donors. A higher BMI-1 expression in CML CD34(+) progenitors was associated with native BMI-1 immune responses. These immune responses to PcG proteins may target leukemia stem cells and have relevance for disease control by GVL.

Hematopoietic stem cells and progenitors of chronic myeloid leukemia express leukemia-associated antigens: implications for the graft-versus-leukemia effect and peptide vaccine-based immunotherapy.

The cure of chronic myeloid leukemia (CML) patients following allogeneic stem cell transplantation (SCT) is attributed to graft-versus-leukemia (GVL) effects targeting alloantigens and/or leukemia-associated antigens (LAA) on leukemia cells. To assess the potential of LAA-peptide vaccines in eliminating leukemia in CML patients, we measured WT1, PR3, ELA2 and PRAME expression in CD34+ progenitor subpopulations in CML patients and compared them with minor histocompatibility antigens (mHAgs) HA1 and SMCY. All CD34+ subpopulations expressed similar levels of mHAgs irrespective of disease phase, suggesting that in the SCT setting, mHAgs are the best target for GVL. Furthermore, WT1 was consistently overexpressed in advanced phase (AdP) CML in all CD34+ subpopulations, and mature progenitors of chronic phase (CP) CML compared to healthy individuals. PRAME overexpression was limited to more mature AdP-CML progenitors only. Conversely, only CP-CML progenitors had PR3 overexpression, suggesting that PR1-peptide vaccines are only appropriate in CP-CML. Surface expression of WT1 protein in the most primitive hematopoietic stem cells in AdP-CML suggest that they could be targets for WT1 peptide-based vaccines, which in combination with PRAME, could additionally improve targeting differentiated progeny, and benefit patients responding suboptimally to tyrosine kinase inhibitors, or enhance GVL effects in SCT patients.

Phenotype and function of a CD56+ peripheral blood monocyte.

G-CSF primed CD34 cells cultured for 2-3 weeks in IL-2 and stem cell factor generate CD56(high) cells with phenotypic and morphologic features of NK cells, and a novel adherent CD56(low) CD16- population expressing myeloid markers (CD33 and HLA-DR). We hypothesized that similar cells might also occur in peripheral blood. In 13/13 normal individuals, we found a circulating population of CD56(low), CD33+, FcgammaRI+, FcgammaRII+, HLA-DR+, CD11b(high), CD14+ monocytes closely resembling the cultured CD56(low)CD33+ cells. They may represent a normal counterpart of the CD56+ CD33+ hybrid myeloid/natural killer cell leukemia. Their mean frequency was 1.3+/-1% (standard deviation), range 0.16-3.5%, of total mononuclear cells. CD56(low)CD33+ cells, primed with cytomegalovirus antigen, induced autologous T-lymphocyte proliferation comparably to CD56-, CD14+ peripheral blood monocytes (PBM). Conversely, CD56(low) cells induced greater T-cell proliferation than CD56- PBM when lymphocyte responders were HLA mismatched. Unstimulated CD56(low)CD33+ cells showed a low antiproliferative effect on K562, which was increased upon LPS stimulation. The pattern of cytokine production by CD56(low)CD33+ cells and PBM largely overlapped; however, they produced detectable levels of IL-6 and IL-1beta. These results define a minor monocyte population with distinct phenotypic and functional features.

Preliminary study of the incidence of disomy in sperm fractions after MicroSort flow cytometry.

Using triple colour fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the disomy level for chromosome 21 and the sex chromosomes in flow cytometric sorted sperm samples. There were no statistically significant differences in the disomy rates when comparing the sorted samples (either for X- or Y-bearing spermatozoa) with non-sorted samples. There were no diploid spermatozoa observed in any sample group after MicroSort processing.

Human live birth and sperm-sex ratios compared.

The human secondary sex ratio is compared with the percentage of Y-chromosome bearing spermatozoa in human semen. Live birth sex ratio is about 51.3%, whereas the overall percentage of Y-chromosome bearing spermatozoa in our study samples was 50.3%, i.e. 1% closer to the proportion expected by Mendelian segregation. The observed difference between live birth and sperm-sex ratios was significant (P < 0.0001). A possible effect of male age on the percentage Y-bearing spermatozoa was found to be non-significant.

Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection.

The world's first deliveries of normal babies after use of flow cytometric separated human sperm cells (MicroSort) for preconception gender selection are reported. Offspring were of the desired female gender in 92.9% of the pregnancies. Most of these pregnancies and births were achieved after simple intrauterine insemination.

Efficiency of MicroSort flow cytometry for producing sperm populations enriched in X- or Y-chromosome haplotypes: a blind trial assessed by double and triple colour fluorescent in-situ hybridization.

Using fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the efficiency of flow cytometry to separate human X- and Y-chromosome bearing spermatozoa. Our data demonstrate that human spermatozoa can be sorted to a purity of 80-90% for X spermatozoa and of 60-70% for Y spermatozoa. Our results using triple FISH fully agree with the sorting treatment used in each case and corroborate the efficiency of the flow sorting technique for sperm sex selection. In these limited samples (200-500 sperm/donor), the frequencies of disomic or diploid spermatozoa were not increased when comparing the sorted samples with unselected samples or with our control series.

Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations.

The discovery of nucleated erythrocytes in maternal circulation provides a potential source for non-invasive prenatal diagnosis. We have evaluated the use of a three-stage procedure to determine the number of cells that are of fetal rather than maternal origin. First, monoclonal antibodies specific for CD45 and CD14 were used in conjunction with a magnetic (MACS) column to deplete unwanted leukocytes from maternal blood. This was followed by a positive MACS enrichment for nucleated erythrocytes, using an anti-CD71 (transferrin receptor) monoclonal antibody. To discriminate between fetal nucleated erythrocytes and those of maternal origin, enriched fractions were simultaneously stained with an anti-fetal haemoglobin (HbF) antibody and hybridized with probes specific for X and Y chromosomes. Samples were then subjected to blind analysis along with negative control samples from non-pregnant volunteers. Using this dual analysis, we were able to determine that less than one nucleated erythrocyte per ml of maternal blood was of fetal origin. Small numbers of these fetal cells were found in 87.5% of pregnancies, ranging from 6 to 35 weeks gestational age. Comparison of HbF and X/Y probe data also suggests that the fetal cells are less suitable for fluorescence in-situ hybridization (FISH) analysis than similar preparations from other sources.

DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease.

We report the world's first clinical pregnancy resulting from DNA-based enrichment for X-bearing human spermatozoa, for prevention of X-linked hydrocephalus. Sperm separation was followed by embryo biopsy and nested multiplex polymerase chain reaction (PCR) for gender determination. Enriched populations of X-bearing spermatozoa ranging from 80 to 89% pure as determined by fluorescence in-situ hybridization (FISH) resulted in in-vitro fertilization (IVF) rates indistinguishable from normal IVF procedures (65%). In two separate biopsy procedures, 7/9 and 15/16 of the resulting embryos were determined to be female by multiplex PCR. Embryo transfer resulted in a karyotypically normal female fetus. This technique should be widely applicable to gender selection for the prevention of genetic disorders.

Gender preselection in humans? Flow cytometric separation of X and Y spermatozoa for the prevention of X-linked diseases.

Human X- and Y-chromosome-bearing spermatozoa were separated based on their DNA content, using modified flow cytometric cell sorting technology. The resulting separation purity of the X-bearing from Y-bearing spermatozoa was evaluated using in-situ hybridization with alpha satellite DNA probes for the X- and Y-chromosomes. In the putative X-enriched-sorted populations, an average of 82% of the spermatozoa showed a hybridization signal with the X probe. Similarly, in the Y-sorted population 75% gave a signal with the Y probe. Sorted X- and Y-bearing spermatozoa were found to maintain their viability for several hours after sorting. These results demonstrate that the human sperm sex ratio can be significantly shifted to favour the selection of female-producing (X) spermatozoa or male-producing (Y) spermatozoa when spermatozoa are flow cytometrically sorted on the basis of DNA content. We propose that flow cytometrically sorted human spermatozoa, used in conjunction with in-vitro fertilization or intra-oviductal insemination, could be used by families who are at risk for X-linked diseases to preferentially produce female offspring. Sorted spermatozoa could also be used to pre-select for male offspring if that were medically indicated.