Multivariate statistical data analysis as a tool to analyze ex vivo expansion dynamics of cytokine-induced killer cells.

BACKGROUND: Cytokine-induced killer (CIK) cells, obtained after mononucleated cell stimulation with interferon-γ, interleukin-2, and anti-CD3 antibody, are constituted by CD3(+) CD56(+) (CIK) cells and a minority of natural killer (NK; CD3(-) CD56(+) ) cells and T-lymphocytes (CD3(+) CD56(-) ) with antitumor effect against hematological malignancies, thus representing a promising immunotherapy strategy. To ensure in vivo antitumor activity it is mandatory to maximize the percentage of CD3(+) 56(+) effector cells, which is highly variable depending on the starting sample and the harvesting day. Based on cytofluorimetric data, we have retrospectively applied multivariate statistical data analysis (MVDA) to 30 expansions building mathematical models able to predict the expansion fate and the optimal CIK harvesting day. METHODS: Cell phenotype was monitored during culture; multivariate batch statistical process control was applied to monitor cell expansion and orthogonal projections to latent structures to predict CIK percentage. RESULTS: Ten expansions had CD3(+) CD56(+) cells ≥ 40% (good batches) and 20 had CD3(+) CD56(+) cells ≤ 40%. In 36.7%, CD3(+) CD56(+) cells reached the highest concentration at day 17 and the others at day 21. We built a highly predictive regression model for estimating CD3(+) CD56(+) cells during culture. Three variables resulted highly informative: NK % at day 0, cytotoxic T-lymphocytes % (CTLs, CD3(+) CD8(+) ) at day 4, and CIK % at day 7. "Good batches" are characterized by a high percentage of CTLs and CD3(+) CD56(+) cells at day 4 and day 7, respectively. CONCLUSION: By applying MVDA it is possible to optimize CIK expansion, deciding the optimal cell harvesting day. A predictive role for CTL and CIK was evidenced.

Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products.

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

BACKGROUND AIMS: A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. METHODS: Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. RESULTS: After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. CONCLUSIONS: The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.

A study on mutual interaction between cytokine induced killer cells and umbilical cord-derived mesenchymal cells: Implication for their in-vivo use.

Recently a number of cellular therapy based-clinical trials have been carried out using mesenchymal stromal cells (MSC) or cytokine-induced-killer (CIK) cells aiming to improve outcome of allogeneic hematopoietic stem cell transplantation. We have isolated MSC from umbilical cord (UC) exploring the interaction between CIK cells and UC-MSC. We found that UC-MSC could suppress CIK cells activity, when co-cultured in a cell-to-cell system. In addition, CIK cells could potentially lyse UC-MSC in a time and ratio dependent manner that could have implications for their in vivo use. Here we provide experimental data on the mutual interaction of CIK cells and UC-MSC, suggesting a negative interference when the two cell types are used in combination. In the light of our observations, when CIK and UC-MSC will be used in clinical trials, timing and sequencing of their infusion should be considered.

Cytosine arabinoside potentiates the apoptotic effect of bendamustine on several B- and T-cell leukemia/lymphoma cells and cell lines.

Bendamustine and cytosine arabinoside (ara-c) are commonly used cytotoxic agents with unique mechanisms of action. We have previously reported a striking additive cytotoxic effect of the consecutive combination of bendamustine and ara-c in mantle cell lymphoma (MCL) cell lines. In the present study, cell lines of follicular lymphoma (DOHH-2), chronic lymphocytic leukemia/lymphoma (EHEB), diffuse large B-cell lymphoma (SU-DHL-4), T-cell leukemia/lymphoma (JURKAT and KARPAS-299) and MCL (JEKO-1 and GRANTA-519) were exposed to the two single drugs or the drugs combined, given simultaneously and consecutively. Peripheral blood chronic lymphocytic leukemia (CLL) B-cells from five patients were also analyzed. Apoptosis, cell proliferation/metabolic activity and mitochondrial damage were evaluated. The combination index (CI) was used to assess synergy between the drugs. Bendamustine exhibited a relevant cytotoxic effect that was dose- and time-dependent, except for SU-DHL-4 and T-cell lymphoma cells. The addition of ara-c after bendamustine significantly potentiated the single-drug cytotoxic effect of bendamustine on all cell lines, including 17p - CLL B-cells, JURKAT and SU-DHL-4, the latter presenting the highest synergism (CI < 0.01). Bendamustine and ara-c are highly synergistic on T- and B-cell lymphoma cells and cell lines, similar to MCL, overcoming resistance to the single agents.

Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene.

The oxygen sensing pathway modulates erythropoietin expression. In normal cells, intracellular oxygen tensions are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins. PHD2 isozyme has a key role in tagging hypoxia-inducible factor (HIF)-α subunits for polyubiquitination and proteasomal degradation. Erythrocytosis-associated PHD2 mutations reduce hydroxylation of HIF-α. The investigation of 67 patients with isolated erythrocytosis, either sporadic or familial, allowed the identification of three novel mutations in the catalytic domain of the PHD2 protein. All new mutations are germ-line, heterozygous and missense, and code for a predicted full length mutant PHD2 protein. Identification of the disease-causing genes will be of critical importance for a better classification of familial and acquired erythrocytosis, offering additional insight into the erythropoietin regulating oxygen sensing pathway.

The cytotoxic effects of bendamustine in combination with cytarabine in mantle cell lymphoma cell lines.

Bendamustine is clinically useful in mantle-cell lymphoma (MCL). Its favorable toxicity profile in-vivo favors its combination with other cytotoxic drugs. Cytarabine is a key drug in the treatment of younger patients with MCL. The current study investigated the in-vitro cytotoxic effect of bendamustine and cytarabine, alone or combined, on two MCL cell lines representing the classic and blastoid variant of the lymphoma subtype (JEKO-1 and GRANTA-519). Cell lines were exposed to each drug alone, or simultaneously and consecutively to both drugs, for different time schedules. Apoptosis was measured by flow cytometry. Mitochondrial damage, cell proliferation/metabolic activity, and cell cycle analysis were also assessed. The synergistic, additive, or antagonistic effects of the drugs were calculated with the combination index (CI) method. Bendamustine and cytarabine alone exhibited relevant cytotoxic activity on both cell lines. Both drugs induced cell cycle arrest in S phase. Drug combinations were associated with significantly higher cytotoxic effects than each drug alone. Among the combination schedules, the consecutive incubation of bendamustine followed by cytarabine was associated with the lower CI, being 10-100-fold lower than with simultaneous incubations. The cytotoxic effect of the consecutive combination was prominent on both cell lines, indicating a very strong and highly significant synergy in inducing apoptosis. Similar results were obtained measuring mitochondrial damage or the decline of the metabolic activity in all cell lines. The strong synergistic effect of bendamustine and cytarabine on MCL cells provides a rationale for developing schedules combining these agents in the treatment of MCL.

Epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-bb can substitute for fetal bovine serum and compete with human platelet-rich plasma in the ex vivo expansion of mesenchymal stromal cells derived from adipose tissue.

BACKGROUND AIMS: Human mesenchymal stromal cells (MSC) are multipotent cells possessing self-renewal capacity, long-term viability and multilineage potential. We analyzed the effect of four different medium supplements on the expansion and differentiation of adipose tissue-derived MSC (ADSC) in order to avoid the use of xenogeneic serum. METHODS: We compared fetal bovine serum (FBS) with 10% human platelet-rich plasma (hPRP), 3% human platelet-poor plasma (hPPP) and with a cytokine cocktail composed of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-bb (PDGFbb) added to 3% hPPP. This mixture was developed testing EGF, bFGF, granulocyte-colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF-I), PDGFbb and transforming growth factor (TGF)-ß1 added alone or in combination with hPPP. RESULTS: Our data demonstrate that the addition of EGF, bFGF and PDGFbb, in a medium supplemented with hPPP, obtainable from 150-200 mL whole autologous blood, supports ADSC expansion better than FBS, as confirmed by cumulative population doublings (cPD; 15.0 ± 0.5 versus 9.4 ± 2.8). The addition of human platelet-rich plasma (hPRP) further improved ADSC proliferation (cPD 20.0 ± 1.2), but the achievement of hPRP presented a major drawback, requiring 1000-1200 mL autologous or donor whole blood. The medium supplements did not influence ADSC phenotype: they expressed CD105, CD90 and CD44 lacking hematopoietic antigens. The exposure to the proposed cocktail or to hPRP increased adipogenic and osteogenic differentiation. CONCLUSIONS: The addition of EGF, bFGF and PDGFbb to hPPP could ensure a sufficient number of ADSC for clinical applications, avoiding the use of animal serum and representing a novel approach in regenerative medicine.

Effect of platelet lysate on the functional and molecular characteristics of mesenchymal stem cells isolated from adipose tissue.

BACKGROUND: Mesenchymal stem cells (MSCs) are non-hematopoietic, adult, fibroblast-like, multipotent cells that are plastic adherent in standard culture conditions. They can be isolated from several tissues, but it is always necessary to expand them for clinical practice. AIM: We investigated the effect of human platelet lysate (hPL) on the expansion of human MSCs isolated from adipose tissue (AT), comparing it with fetal bovine serum (FBS) and human platelet-poor plasma (hPPP). MATERIALS AND METHODS: Human AT-MSCs, hPL and hPPP were obtained from 7 healthy subjects. AT-MSCs were seeded at 1500 cells/cm(2) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 10% hPPP or 10% hPL. Cells were harvested, counted and analyzed by flow cytometry every 7 days for 5 passages (P). The differentiation assays, RNA isolation and co-culture with allogeneic lymphocytes were performed at the end of P2. RESULTS: AT-MSCs achieved a better proliferation rate when cultured with hPL than with hPPP or FBS (20 ±2 versus 8 ±3 and 6 ±3, respectively, at the end of P5 [p<0.01]). hPL preserved the differentiation capacity and typical expression of surface antigens, avoiding the risks associated with the use of animal derivatives. AT-MSCs demonstrated a stronger inhibitory effect on lymphocyte proliferation with hPL than with other culture conditions, even at a AT-MSCs:T cells ratio of 1:10. The transcriptional level of matrix metalloproteinase 2, used to evaluate stemness, was very high in all conditions tested. CONCLUSIONS: hPL represents an effective and safe supplement for MSC expansion to be used in the clinical setting.

Identical IGHV-D-J gene rearrangement may precede the clinical onset of chronic lymphocytic leukemia by several years.

The pathogenesis of chronic lymphocytic leukemia (CLL) has not been fully elucidated. Moreover, the time required for the initial B lymphocyte IGH gene rearranged clone to manifest as a clinical entity remains unknown. We searched for previous IGH gene rearranged B lymphocyte clones in healthy people who developed CLL and estimated the time for the clone to become clinically detectable. To this aim, we identified all incident cases of CLL diagnosed in a cohort of 15,055 healthy subjects aged 18-65 years enrolled in a prospective survey on thrombophilia. Seven CLL cases were identified at a median follow-up of 54 months (range, 18-89). The estimated incidence was 0.46 cases/10,000 person-years (CI: 0.17-1.00). A PCR was performed to detect IGH gene rearrangement at enrollment and at CLL diagnosis. Comparison was possible in six subjects. In five, the same IGH gene rearrangement and gene sequence were already present 39-89 months before CLL diagnosis. A mutated status was identified in four of five cases. The median age at diagnosis was 66.2 years, and all subjects were asymptomatic. Two patients expressing the IGHV1-69 gene with an unmutated status required treatment 16 and 40 months after diagnosis. The IGHV4 family genes were rearranged in the remaining cases, all showing a mutated status. No additional rearrangements or mutations in the rearranged gene were found during follow-up. An identical clonal IGH gene rearrangement may precede CLL diagnosis by several years.

Loss of the JAK2 intramolecular auto-inhibition mechanism is predicted by structural modelling of a novel exon 12 insertion mutation in a case of idiopathic erythrocytosis.

We report a novel gain-of-function JAK2 exon 12 insertion mutation in a patient with idiopathic erythrocytosis and low serum erythropoietin level. To date, only rare cases of such mutations have been reported in the JAK2 exon 12. Using computer-based structural modelling we propose that this mutation causes the loss of the JAK2 auto-inhibition step, leading to the constitutive activation of JAK2 tyrosine kinase-dependent activity. Our model-based hypothesis provides a useful approach for the investigation of the phenotype-genotype relationship in myeloproliferative disorders involving JAK2.

Impact of immune thrombocytopenia on the clinical course of chronic lymphocytic leukemia.

The prevalence, clinical characteristics, and prognostic significance of immune thrombocytopenia (IT) in patients with chronic lymphocytic leukemia (CLL) have not been clearly determined. To clarify this, we retrospectively analyzed 1278 consecutive newly diagnosed patients with CLL. Criteria for IT diagnosis included the following: rapid (< 2 weeks) and severe fall (half of the initial level and below 100 x 10(9)/L) in platelet count; normal or augmented megakaryocytes in bone marrow; no or limited (not palpable) splenomegaly; no cytotoxic treatment in the preceding month. Sixty-four patients (5%) were diagnosed with IT. The median time to IT from CLL diagnosis was 13 months (range, 0-81 months), and median platelet count at IT diagnosis was 14 x 10(9)/L (range, 1-71 x 10(9)/L). Fifty-six of the 64 patients (87%) received treatment for IT. The probability of responding to treatment for IT was significantly higher for patients receiving chemotherapy with or without steroids than for patients treated with intravenous immunoglobulins with or without steroids (P = .01). The development of IT was significantly associated with unmutated IgVh, a positive direct antiglobulin test, and the occurrence of autoimmune hemolytic anemia. Patients with CLL and IT had poorer survival than other patients with CLL (5-year overall survival 64% vs 82%, P < .001), and this effect was independent from common clinical prognostic variables.

Flow cytometry in the diagnosis of drug-induced thrombocytopenia: two illustrative cases.

Drug-induced thrombocytopenia is a challenging diagnosis in the clinical practice because of the many drugs or alternative causes that may be implicated. Exact identification of such drug(s) is required to guide future management and avoid re-exposure. We describe two cases of isolated thrombocytopenia in which cytometric analysis, a readily available technique, allowed the identification of the causative drug in the context of complex therapies (rifampicin and abciximab causing late onset thrombocytopenia).

Intraindividual consistency of the activated protein C resistance phenotype.

Resistance to activated protein C (APC) has been demonstrated to be a risk factor for venous thromboembolism, but it is not known whether this phenotype is consistent over time. We reinvestigated 2580 subjects from the Vicenza Thrombophilia and Atherosclerosis (VITA) Project to evaluate the prevalence of a consistent APC resistance phenotype in the population. Among the 433 subjects with an APC resistance at first visit, the phenotype was confirmed in all the 74 factor V (FV) Leiden carriers and in 124 of 359 FV Leiden negative subjects (34%). The prevalence of a confirmed phenotype, not associated with FV Leiden, was 4.8% in our population. In a subgroup of subjects previously investigated for heritability of the APC resistance, we confirmed the APC resistance phenotype in seven of 39 (17.9%) subjects with an APC resistant sibling but only in 20 of 408 (4.9%) subjects without a sibling with the same phenotype (P = 0.005). Among the 124 FV Leiden negative subjects with a persistent APC resistance phenotype, 40 (32%) had a plasma factor VIII coagulant activity level above 150 IU/dl and eight (6.4%) were carriers of the G20210A prothrombin allele. APC resistance not due to FV Leiden is a frequent and consistent phenotype in the general population, with a possibly strong genetic influence.

Multiplex amplification and fluorimetric detection of short tandem repeats for mixed chimerism after bone marrow transplant.

We present the clinical application of a simple method for mixed chimerism analysis after allogeneic bone marrow (BM) or peripheral blood stem cell (PBSC) transplant. A commercial kit which enables multiplex amplification of 9 highly polymorphic short tandem repeats in a single reaction tube was used. Molecular size and relative quantities of each amplified fragment are subsequently measured using an automated fluorimeter. The sensitivity and linearity were tested using whole blood or genomic DNA from two subjects, mixed in various proportions from 0.25% to 99.75%. In 70 donor-recipient pairs the median number of informative alleles for the assessment of relapse was 5.9 (range 3-11). Results showed that the linearity between the measured and expected relative quantities of amplified DNA showed a regression coefficient of 0.99 in the interval 10-90%. The mean sensitivity was 1.5% (range 0.5-2.5), greater than previously reported. A total of 70 cases of allogeneic transplant (49 with family and 21 with unrelated donors) were monitored before transplant and after 1, 2, 4, 6 or 12 months thereafter and then at 6 months intervals (range 6-36). In 18 patients mixed chimerism was observed 1 month from transplant, with donor allele percentage ranging from 1 to 6%. Fragment dimension reproducibility (CV 0.34, range 0.52-0.66) was confirmed by an internal DNA control and by amelogenin fragment length repeatability on all patients. In conclusion, the proposed method is sufficiently simple, rapid, sensible, specific and cost effective for the evaluation of mixed chimerism after BM or PBSC transplant in a clinical setting.

Fluorescent polymerase chain reaction and capillary electrophoresis for IgH rearrangement and minimal residual disease evaluation in multiple myeloma.

BACKGROUND AND OBJECTIVES: Several polymerase chain reaction (PCR)-based techniques for tracking minimal residual disease (MRD) in B-lymphoproliferative disorders have been recently proposed. These procedures show significant variation in sensitivity and specificity. We describe an alternative assay based on fluorescent PCR combined with capillary electrophoresis and GeneScan analysis, to identify the monoclonal immunoglobulin heavy chain (IgH) rearrangement in multiple myeloma (MM) and to provide a semi-quantitative evaluation of MRD by limiting dilutions. DESIGN AND METHODS: Different sets of family specific primers derived from the leader region and from the framework-1 of IgH were used, with a unique reverse fluorescent primer JH. The malignant clone was identified by GeneScan and sequenced. Two tumor primers, mapping in the complementarity determining regions CDRII and CDRIII, were designed for each patient. A comparison between the nested-PCR approach and direct fluorescent PCR was performed for three patients in complete clinical remission after autologous or allogeneic bone marrow transplantation. RESULTS: Thirty-six consecutive patients with MM were screened and monoclonality was identified in about 70% of the cases. Molecular MRD evaluation was performed in 18 patients using tumor primers. This method allowed identification of 1 neoplastic cell among 10(4)-10(6) normal cells. In three cases, negative by nested-PCR and agarose gel electrophoresis, gene scanning showed persistence of the neoplastic clone, despite the negativity of the immunofixation. INTERPRETATION AND CONCLUSIONS: Capillary electrophoresis of fluorescent fragments with gene scanning provides a simple, rapid and reproducible method to detect IgH rearrangement and to evaluate MRD. Furthermore, the sensitivity reached is up to 1 log higher than that of the conventional approach with nested-PCR, even though two steps of specificity are maintained.