Retroviral gene transfer to repopulating hematopoietic cells

Hematopoietic stem cells (HSCs) have the capacity to maintain hematopoiesis throughout life. HSCs are ideal targets for permanent gene transfer, as the transgene will be expressed in all the progeny of the gene-modified stem cells. The aim of this thesis was to optimize conditions for retroviral gene transfer to human candidate HSCs and to study mechanisms interfering with efficient gene transfer. First, long-term bone marrow culture transduction of murine hematopoietic cells was investigated, showing inefficient gene transfer and loss of repopulating ability of the transduced cells. Thereafter, transduction of human CD34+ cells was studied using the MGIN vector with the GALV, amphotropic and VSV-G envelopes. Amphotropic, and, particularly, GALV vector containing medium (VCM) inhibited transduction on fibronectin preloaded with vector. GALV VCM inhibited transduction of NOD/SCID repopulating cells (SRCs) as well. Optimal transduction was seen with vector preloaded to fibronectin and cells added in medium without vector. To study the mechanisms underlying GALV vector mediated inhibition, the GLVR1 receptor was overexpressed using phorbol ester or a retroviral vector, showing complete abolishment the inhibitory effect from GALV VCM. Together our results show that low levels of GLVR1, in combination with non-infectious vector particles in VCM, limit transduction of hematopoietic progenitors. Finally, vectors with GALV, amphotropic and VSV-G envelopes were compared regarding their ability to transfer genes to SRCs. The different pseudotypes were equally efficient in the presence of serum with an average transduction efficiency of 25-33 % of SRCs, but the engraftment levels were reduced compared to fresh cells. Only the GALV vectors could be used under serum-free conditions showing high level transduction of SRCs (46 %) without loss of SRC frequency as analyzed by transplantation at limiting cell numbers. In conclusion, under optimal conditions, highly efficient gene transfer human hematopoietic cells with NOD/SCID repopulating ability could be accomplished. However, for clinical applications, safety issues, particularly regarding the risk of mutagenesis from retroviral vector insertion into the genome of repopulating cells, need to be adressed carefully

Treatment outcome in T-cell lymphoblastic lymphoma in adults - a population-based study from the Swedish Lymphoma Registry

Background. T-cell lymphoblastic lymphoma (T-LBL) is a rare neoplasm of precursor lymphoblast origin, for which there is no standard treatment for adults. Results of current treatment strategies in selected populations do exist but are largely unreported for unselected series. Here, we aimed to investigate treatment outcome in a population-based cohort. Material and methods. Patients were identified through the Swedish Lymphoma Registry and data was retrospectively collected for all adult (>= 18 years) Swedish T-LBL patients diagnosed during 2000-2009. Results. A total of 39 patients with median age 40 years (range 18-78) were identified with females being significantly older than males (median age 66 vs. 37, p = 0.027). The five-year overall survival for all patients was 42%. Female gender was associated with shorter survival also when adjusted for treatment strategy and age [hazard ratio (HR) 4.29; p = 0.002]. Thirty patients received intensive chemotherapy, otherwise used for treatment of acute lymphoblastic leukemia (ALL), which resulted in an overall response rate of 97% and a five-year progression-free survival (PFS) of 49%. In this group only CNS involvement at diagnosis predicted shorter PFS (HR 13.3; p = 0.03). Among patients treated with hyper-CVAD the addition of mediastinal irradiation resulted in prolonged time to progression compared to patients receiving only chemotherapy (p = 0.047). The major reason for treatment failure was relapse and in this series 18-fluoro-deoxyglucose positron emission tomography (PET) did not predict this risk. Conclusion. This population-based study indicates that all fit T-LBL patients should be considered for intensive treatment. Our results also suggest a beneficial effect of mediastinal irradiation in combination with hyper-CVAD treatment. Relapsing patients have a dismal outcome irrespective of salvage treatment

Hudlymfom – ovanlig sjukdom ofta med lång tid till diagnos

Primary cutaneous lymphoma is a heterogeneous group of diseases where the malignant lymphocytes are primarily present in the skin at the time of diagnosis. The most common type of primary cutaneous lymphoma is mycosis fungoides. Early stages of mycosis fungoides present with flat or slightly elevated red skin lesions and can resemble eczema or psoriasis. In advanced stages erythrodermia or skin tumors can develop. For many patients with mycosis fungoides effective albeit not curative treatment is available. Large randomized treatment studies for mycosis fungoides are largely lacking, which makes decisions on treatment strategy difficult. The new national clinical guidelines will hopefully enable more equal care for patients with mycosis fungoides and other types of primary cutaneous lymphoma in Sweden

Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes

Background The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34(+)/CD38(low) and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes. Methods CB cells were transduced on Retronectin using an MSCV-based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13-MGIN (GALV) 293GPG-MGIN (VSV-G) or AM12-MGIN (amphotropic). Results Sorted CD34(+)/CD38(low) cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP-positive cells was 61.8+/-6.6% (PG13-MGIN), 26.9+/-3.5% (293GPG-MGIN), and 39.3+/-4.8% (AM12-MGIN). For transplantation experiments, CD34(+) cells were prestimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5 x 10(5) cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum-free medium and transduction of CD34(+) cells using VSV-G-pseudotyped vectors under serum-free conditions was very inefficient. In contrast, transduction with PG13-MGIN under serum-free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3+/-6.6%), and efficient gene transfer to SRCs (46.2+/-4.8%). Conclusions The best conditions for transduction and engraftment of CB SRCs were obtained with GALV-pseudo typed vectors using serum-free conditions. Copyright (C) 2002 John Wiley Sons, Ltd

The histone deacetylase inhibitor valproic acid sensitizes diffuse large B-cell lymphoma cell lines to CHOP-induced cell death.

Epigenetic code modifications by histone deacetylase inhibitors (HDACis) have recently been proposed as potential new therapies for hematological malignancies. Diffuse large B-cell lymphoma (DLBCL) is the most common form of aggressive lymphoma. At present, standard first line treatment for DLBCL patients is the antracycline-based chemotherapy regimen CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) combined with the monoclonal anti-CD20 antibody rituximab (R-CHOP). Since only 50-60% of patients reach a long-time cure by this treatment, there is an urgent need for novel treatment strategies to increase the response and long-term remission to initial R-CHOP therapy. In this study, we investigated the effect of the HDAC inhibitor valproic acid (VPA) on DLBCL cell lines. To elucidate the effects of VPA on chemo-sensitivity, we used a cell-line based model of CHOP-refractory DLBCL. All five DLBCL cell lines treated with VPA alone or in combination with CHOP showed decreased viability and proliferation. The VPA-induced sensitization of DLBCL cells to cytotoxic treatment resulted in increased number of apoptotic cell as judged by annexin V-positivity and the presence of cleaved caspase-3. In addition, pretreatment with VPA resulted in a significantly increased DNA-damage as compared to CHOP alone. In summary, HDAC inhibitors such as VPA, are promising therapeutic agents in combination with R-CHOP for patients with DLBCL

Central nervous system relapse in peripheral T-cell lymphomas: A Swedish lymphoma registry study.

Central Nervous System (CNS) relapse in non-Hodgkin lymphomas (NHL) carries a very poor prognosis. Risk factors and outcome have been studied in aggressive B-cell lymphomas but very little is known about the risk in peripheral T-cell lymphoma (PTCL). We aimed at analyzing risk factors for CNS involvement at first relapse or progression, and the outcome of these patients, in a large population-based cohort of PTCL patients. Twenty-eight out of 625 patients (4.5%) developed CNS disease over time. In multivariable analysis disease characteristics at diagnosis independently associated with an increased risk of later CNS involvement were involvement of >1 extranodal site (Hazard Ratio [HR] 2.60, 95% Confidence Interval [95% CI] 1.07-6.29, p=0.035), skin (HR 3.51, 95% CI 1.26-9.74, p=0.016) and gastrointestinal involvement (HR 3.06, 95% CI 1.30-7.18, p=0.010). The outcome of relapsed/refractory patients was very poor and CNS involvement was not associated with a significantly worse outcome compared to relapsed/refractory patients without CNS involvement in multivariable analysis (HR 1.6, 95% CI 0.96-2.6, p=0.074). The results from the present study indicate that CNS relapse in PTCL occurs at a frequency similar to what is seen in aggressive B-cell lymphomas, but the poor outcomes in relapse are largely driven by systemic rather than CNS disease

Real world data on prognostic factors and treatment in peripheral T-cell lymphomas: a study from the Swedish Lymphoma Registry.

Peripheral T-cell Lymphomas (PTCLs) are rare lymphomas with mostly poor outcome with current treatment. Addition of etoposide to CHOP and up-front consolidation with autologous stem cell transplantation (autoSCT) have shown promising results, but have never been tested in randomized trials. As a complement to retrospective analyses of clinical trials, we aimed at analyzing prognostic factors and outcome in an unselected, population-based cohort. Through the Swedish Lymphoma Registry we identified 755 PTCL patients diagnosed during a 10-year period. In addition to International Prognostic Index (IPI) factors, male gender was associated with an adverse overall survival (OS) (HR 1.28, p=0.011) and progression-free survival (PFS) (HR 1.26, p=0.014). In an intention-to-treat analysis in 252 nodal PTCL and EATL patients (excluding ALK-positive ALCL), up-front autoSCT was associated with a superior OS (HR 0.58, p=0.004) and PFS (HR 0.56, p=0.002) compared to patients treated without autoSCT. Addition of etoposide to CHOP resulted in superior PFS in patients up to 60 years (HR 0.49, p=0.008). This study is the largest population-based PTCL cohort reported so far and provides important information on outcome in PTCL outside the setting of clinical trials

Low level of gene transfer to and engraftment of murine bone marrow cells from long-term bone marrow cultures

OBJECTIVE: We wanted to determine whether the long-term bone marrow culture (LTBMC) transduction system would lead to efficient gene transfer and engraftment of murine repopulating hematopoietic stem cells (HSC), particularly in nonablated recipients. MATERIALS AND METHODS: Congenic mouse strains expressing Ly 5.1 or Ly 5.2 and the GP+E86 cell line producing the MGirL22Y vector carrying the gene for enhanced GFP were used. Murine LTBMCs were established and demi-depopulated on days 7 and 14 with addition of vector supernatant on days 8 and 15. RESULTS: Cell recovery on day 21 was 21.3%+/-3.8% of input cells and CFU-C recovery was 9.7+/-3.4% as compared with CFU-C of input cells. In vitro transduction efficiency determined by CFU-C expressing GFP was 22.2%+/-1.6%. In irradiated (950 cGy) mice transplanted with 2x10(6) LTBMC cells, 94% of nucleated cells in the blood at week 16 were of donor origin. However, GFP was only detected at low level in a few animals at week 4 and not later. Analysis of bone marrow from these mice at week 20 did not show any GFP expression and semiquantitative PCR revealed a transgene level of <1%. When 3.5-20.8x10(6) LTBMC cells (corresponding to 20-100x10(6) fresh cells) were transplanted to nonablated recipients, no engraftment or GFP expression were detected. Competitive repopulation experiments showed that the long-term repopulation ability (LTRA) of the LTMC cells was only 7% of fresh cells. CONCLUSION: These results indicate that LTBMC transduction of murine cells leads to low-level transduction of progenitors, no gene transfer to repopulating stem cells, and reduction in LTRA in ablated and nonablated recipients

Mycosis Fungoides: A Retrospective Study of 44 Swedish Cases.

Mycosis fungoides (MF) is a primary cutaneous T-cell lymphoma with slow disease progression. There is a lack of descriptive data from Sweden concerning patients with this diagnosis. This study extracted data on patients admitted to the dermatology department at Lund University Hospital, Sweden from 1996 to 2010. Forty-four patients with clinically and histopathologically verified MF were identified during the period, with a mean follow-up time of 5.6 years. Median age at initial diagnosis was 64 years. In several cases other skin diseases preceded MF onset, such as non-specific dermatitis (32%) and parapsoriasis (30%). The majority of patients (86%, n = 38) had limited-stage (IA-IB) disease at the time of diagnosis. Overall response rate to psoralen plus ultraviolet A (PUVA) treatment was 81%. In adnexal MF, a trend to higher rate of progression to an advanced stage was observed when compared with non-adnexal disease (40% and 21%, respectively). Increased levels of soluble interleukin-2 (IL-2) receptor correlated with disease stage, being elevated in advanced stages or adnexal disease, but almost never in early non-adnexal limited-stage disease. Overall mortality was 25%, but only 11% could be verified as caused by MF