Prognostic and predictive impact of metastatic organ involvement on maintenance therapy in advanced metastatic colorectal cancer: Subgroup analysis of patients treated within the PanaMa trial (AIO KRK 0212).

Despite molecular selection, patients (pts) with RAS wildtype mCRC represent a heterogeneous population including diversity in metastatic spread. We investigated metastatic patterns for their prognostic and predictive impact on maintenance therapy with 5-fluorouracil/folinic acid ± panitumumab. The study population was stratified according to (1) number of involved metastatic sites (single vs multiple organ metastasis), liver-limited disease vs (2) liver metastasis plus one additional site, and (3) vs liver metastasis plus ≥two additional sites. Kaplan-Meier method and Cox regressions were used to correlate efficacy endpoints. Single organ metastasis was observed in 133 pts (53.6%) with 102 pts (41.1%) presenting with liver-limited disease, while multiple organ metastases were reported in 114 pts (46.0). Multiple compared to single organ metastases were associated with less favorable PFS (HR 1.48, 95% CI 1.13-1.93; P = .004) and OS (HR 1.37, 95% CI 0.98-1.93; P = .068) of maintenance therapy. While metastatic spread involving one additional extrahepatic site was not associated with clearly impaired survival compared to liver-limited disease, pts with liver metastasis plus ≥two additional sites demonstrated less favorable PFS (HR 1.92, 95% CI 1.30-2.83; P < .001), and OS (HR 2.38, 95% CI 1.51-3.76; P < .001) of maintenance therapy. Pmab-containing maintenance therapy appeared active in both pts with multiple (HR 0.58; 95% CI, 0.39-0.86; P = .006) as well as to a lesser numerical extent in pts with single organ metastasis (HR 0.83; 95% CI, 0.57-1.21; P = .332; Interaction P = .183). These data may support clinical decisions when EGFR-based maintenance therapy is considered.

Negative Hyperselection of Resistance Mutations for Panitumumab Maintenance in RAS Wild-Type Metastatic Colorectal Cancer (PanaMa Phase II Trial, AIO KRK 0212).

PURPOSE: We evaluated additional mutations in RAS wild-type (WT) metastatic colorectal cancer (mCRC) as prognostic and predictive biomarkers for the efficacy of added panitumumab to a 5-fluorouracil plus folinic acid (FU/FA) maintenance as pre-specified analysis of the randomized PanaMa trial. PATIENTS AND METHODS: Mutations (MUT) were identified using targeted next-generation sequencing (NGS; Illumina Cancer Hotspot Panel v2) and IHC. RAS/BRAF V600E/PIK3CA/AKT1/ALK1/ERBB2/PTEN MUT and HER2/neu overexpressions were negatively hyperselected and correlated with median progression-free survival (PFS) and overall survival (OS) since start of maintenance treatment, and objective response rates (ORR). Univariate/multivariate Cox regression estimated hazard ratios (HR) and 95% confidence intervals (CI). RESULTS: 202 of 248 patients (81.5%) of the full analysis set (FAS) had available NGS data: hyperselection WT, 162 (80.2%); MUT, 40 (19.8%). From start of maintenance therapy, hyperselection WT tumors were associated with longer median PFS as compared with hyperselection MUT mCRC (7.5 vs. 5.4 months; HR, 0.75; 95% CI, 0.52-1.07; P = 0.11), OS (28.7 vs. 22.2 months; HR, 0.53; 95% CI, 0.36-0.77; P = 0.001), and higher ORR (35.8% vs. 25.0%, P = 0.26). The addition of panitumumab to maintenance was associated with significant benefit in hyperselection WT tumors for PFS (9.2 vs. 6.0 months; HR, 0.66; 95% CI, 0.47-0.93; P = 0.02) and numerically also for OS (36.9 vs. 24.9 months; HR, 0.91; 95% CI, 0.61-1.36; P = 0.50), but not in hyperselection MUT tumors. Hyperselection status interacted with maintenance treatment arms in terms of PFS (P = 0.06) and OS (P = 0.009). CONCLUSIONS: Extended molecular profiling beyond RAS may have the potential to improve the patient selection for anti-EGFR containing maintenance regimens.

Individual quality assessment of autografting by probability estimation for clinical endpoints: a prospective validation study from the European group for blood and marrow transplantation.

The aim of supportive autografting is to reduce the side effects from stem cell transplantation and avoid procedure-related health disadvantages for patients at the lowest possible cost and resource expenditure. Economic evaluation of health care is becoming increasingly important. We report clinical and laboratory data collected from 397 consecutive adult patients (173 non-Hodgkin lymphoma, 30 Hodgkin lymphoma, 160 multiple myeloma, 7 autoimmune diseases, and 28 acute leukemia) who underwent their first autologous peripheral blood stem cell transplantation (PBSCT). We considered primary endpoints evaluating health economic efficacy (eg, antibiotic administration, transfusion of blood components, and time in hospital), secondary endpoints evaluating toxicity (in accordance with Common Toxicity Criteria), and tertiary endpoints evaluating safety (ie, the risk of regimen-related death or disease progression within the first year after PBSCT). A time-dependent grading of efficacy is proposed with day 21 for multiple myeloma and day 25 for the other disease categories (depending on the length of the conditioning regimen) as the acceptable maximum time in hospital, which together with antibiotics, antifungal, or transfusion therapy delineates four groups: favorable (≤7 days on antibiotics and no transfusions; ≤21 [25] days in hospital), intermediate (from 7 to 10 days on antibiotics and <3 transfusions, ≤21 to 25 days in hospital or ≥7 days on antibiotics and no transfusions; from 21 to 30 days [25 to 34] in hospital), unfavorable (>7 days on antibiotics, >3 but <6 transfusions; >30/34 days in hospital after transplantation), and very unfavorable (>10 days on antibiotics, >6 transfusions; >30 to 34 days in hospital). The multivariate analysis showed that (1) PBSC harvests of ≥4 × 10(6)/kg CD34 + cells in 1 apheresis procedure were associated with a favorable outcome in all patient categories except acute myelogenous leukemia and acute lymphoblastic leukemia (P = .001), (2) ≥5 × 10(6)/kg CD34 + cells infused predicted better transplantation outcome in all patient categories (P < .0001) except acute myelogenous leukemia and acute lymphoblastic leukemia, (3) 1 or 2 aphereses (P = .001) predicted good outcome, (4) toxicity increased with higher graft volume reinfused (>500 mL) (P = .002), and (5) patients with a central venous catheter during both collection and infusion of PBSC had a more favorable outcome post-PBSCT than peripheral access (P = .007). The type of mobilization regimen did not affect the outcome of auto-PBSCT. The present study identified predictive variables, which may be useful in future individual pretransplantation probability evaluations with the goal to improve supportive care.

Study design: two long-term observational studies of the biosimilar filgrastim Nivestim™ (Hospira filgrastim) in the treatment and prevention of chemotherapy-induced neutropenia.

BACKGROUND: Nivestim™ (filgrastim) is a follow-on biologic agent licensed in the EU for the treatment of neutropenia and febrile neutropenia induced by myelosuppressive chemotherapy. Nivestim™ has been studied in phase 2 and 3 clinical trials where its efficacy and safety was found to be similar to its reference product, Neupogen®. Follow-on biologics continue to be scrutinised for safety. We present a design for two observational phase IV studies that are evaluating the safety profile of Nivestim™ for the prevention and treatment of febrile neutropenia (FN) in patients treated with cytotoxic chemotherapy in general clinical practice. METHODS/DESIGN: The NEXT (Tolérance de Nivestim chez les patiEnts traités par une chimiothérapie anticancéreuse cytotoXique en praTique courante) and VENICE (VErträglichkeit von NIvestim unter zytotoxischer Chemotherapie in der Behandlung malinger Erkrankungen) trials are multicentre, prospective, longitudinal, observational studies evaluating the safety profile of Nivestim™ in 'real-world' clinical practice. Inclusion criteria include patients undergoing cytotoxic chemotherapy for malignancy and receiving Nivestim as primary or secondary prophylaxis (NEXT and VENICE), or as treatment for ongoing FN (NEXT only). In accordance with European Union pharmacovigilance guidelines, the primary objective is to evaluate the safety of Nivestim™ by gathering data on adverse events in all system organ classes. Secondary objectives include obtaining information on patient characteristics, efficacy of Nivestim™ therapy (including chemotherapy dose intensity), patterns of use of Nivestim™, and physician knowledge regarding filgrastim prescription and the reasons for choosing Nivestim™. Data will be gathered at three visits: 1. At the initial inclusion visit, 2. At a 1-month follow-up visit, and 3. At the end of chemotherapy.Recruitment for VENICE commenced in July 2011 and in November 2011 for NEXT. VENICE completed recruitment in July 2013 with 407 patients, and NEXT in September 2013 with 2123 patients. Last patient, last visit for each study will be December 2013 and March 2014 respectively. DISCUSSION: The NEXT and VENICE studies will provide long-term safety, efficacy and practice pattern data in patients receiving Nivestim™ to support myelosuppressive chemotherapy in real world clinical practice. These data will improve our understanding of the performance of Nivestim™ in patients encountered in the general patient population. TRIAL REGISTRATION: NEXT NCT01574235, VENICE NCT01627990.

Current clinical indications for plerixafor.

Autologous and allogeneic hematopoietic stem cell (HSC) transplantation are considered the standard of care for many malignancies including lymphoma, multiple myeloma, and some leukemias. In many cases, mobilized peripheral blood has become the preferred source for HSCs. Plerixafor, an inhibitor of the interaction between CX chemokine receptor 4 (CXCR4) and stromal derived factor-1 alpha (SDF-1), has been evaluated in clinical trials and approved by the FDA and EMA. This agent has very modest toxicity and appears to be quite potent at HSC mobilization. Current clinical indications for the use of plerixafor are the subject of this review.

Health-related quality of life in patients with RAS wild-type metastatic colorectal cancer treated with fluorouracil and folinic acid with or without panitumumab as maintenance therapy: a prespecified secondary analysis of the PanaMa (AIO KRK 0212) trial.

BACKGROUND: The PanaMa trial demonstrated significant benefit in progression-free survival with the addition of panitumumab (Pmab) to fluorouracil and folinic acid (FU/FA) as maintenance therapy following first-line induction therapy with FOLFOX/Pmab in patients with RAS wild-type metastatic colorectal cancer. Here, we report health-related quality of life (HRQOL) analyses from the PanaMa trial. METHODS: HRQOL outcomes were evaluated using European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) at every cycle of therapy until disease progression/death. HRQOL outcomes were mean and individual changes in EORTC QLQ-C30 from baselines (before induction therapy and before maintenance therapy) to each cycle of treatment. Comparative analyses were performed by randomisation status and treatment arm for induction- and maintenance-therapy, respectively. The trial is registered with clinicaltrials.gov (NCT01991873). RESULTS: At least one HRQOL questionnaire was completed by a total of 349/377 (93%) patients who received induction therapy, and by 237/248 (96%) patients who were randomised and received maintenance therapy. During induction therapy, most HRQOL dimensions remained stable or showed improvement, while appetite loss and diarrhoea significantly deteriorated. During maintenance therapy, HRQOL dimensions remained stable, while those that deteriorated during induction therapy showed significant improvement, without significant differences between the treatment arms. CONCLUSION: Maintenance therapy improves HRQOL dimensions that initially deteriorated during induction therapy while stabilising HRQOL in other dimensions. The addition of Pmab to FU/FA as maintenance therapy in patients with RAS wild-type metastatic colorectal cancer prolongs progression-free survival without negative impact on HRQOL.

Depth of response of induction therapy and consecutive maintenance treatment in patients with RAS wild-type metastatic colorectal cancer: An analysis of the PanaMa trial (AIO KRK 0212).

BACKGROUND: In patients with RAS wild-type metastatic colorectal cancer, depth of response (DpR) has gained importance as a novel end-point in clinical trials. We investigated the overall DpR, as well as the prognostic and predictive impact of DpR to induction therapy (six cycles of 5-fluorouracil, leucovorin [FU/FA], oxaliplatin [FOLFOX] and panitumumab [Pmab]) on consecutive maintenance therapy (FU/FA plus Pmab or FU/FA alone) in patients treated within the PanaMa trial. METHODS: Central radiological assessment was performed according to RECIST 1.1. DpR was defined as percentage change in tumour diameter within defined time intervals (induction therapy, maintenance therapy, total course of therapy). For prognostic and predictive analyses, median DpR (

Consensus Molecular Subtypes as Biomarkers of Fluorouracil and Folinic Acid Maintenance Therapy With or Without Panitumumab in RAS Wild-Type Metastatic Colorectal Cancer (PanaMa, AIO KRK 0212).

PURPOSE: Consensus molecular subtypes (CMSs) were evaluated as prognostic and predictive biomarkers of patients with RAS wild-type metastatic colorectal cancer (mCRC) receiving fluorouracil and folinic acid (FU/FA) with or without panitumumab (Pmab) after Pmab + mFOLFOX6 induction within the randomized phase II PanaMa trial. METHODS: CMSs were determined in the safety set (ie, patients that received induction) and full analysis set (FAS; ie, randomly assigned patients who received maintenance) and correlated with median progression-free survival (PFS) and overall survival (OS) since the start of induction or maintenance treatment and objective response rates (ORRs). Hazard ratios (HRs) and 95% CI were calculated by univariate/multivariate Cox regression analyses. RESULTS: Of 377 patients of the safety set, 296 (78.5%) had available CMS data: CMS1/2/3/4: 29 (9.8%)/122 (41.2%)/33 (11.2%)/112 (37.8%) and unclassifiable: 17 (5.7%). The CMSs were prognostic biomarkers in terms of PFS (P < .0001), OS (P < .0001), and ORR (P = .02) since the start of induction treatment. In FAS patients (n = 196), with CMS2/4 tumors, the addition of Pmab to FU/FA maintenance therapy was associated with longer PFS (CMS2: HR, 0.58 [95% CI, 0.36 to 0.95], P = .03; CMS4: HR, 0.63 [95% CI, 0.38 to 1.03], P = .07) and OS (CMS2: HR, 0.88 [95% CI, 0.52 to 1.52], P = .66; CMS4: HR, 0.54 [95% CI, 0.30 to 0.96], P = .04). The CMS interacted significantly with treatment in terms of PFS (CMS2 v CMS1/3: P = .02; CMS4 v CMS1/3: P = .03) and OS (CMS2 v CMS1/3: P = .03; CMS4 v CMS1/3: P < .001). CONCLUSION: The CMS had a prognostic impact on PFS, OS, and ORR in RAS wild-type mCRC. In PanaMa, Pmab + FU/FA maintenance was associated with beneficial outcomes in CMS2/4, whereas no benefit was observed in CMS1/3 tumors.

Panitumumab Plus Fluorouracil and Folinic Acid Versus Fluorouracil and Folinic Acid Alone as Maintenance Therapy in RAS Wild-Type Metastatic Colorectal Cancer: The Randomized PANAMA Trial (AIO KRK 0212).

PURPOSE: The randomized PANAMA trial investigated the efficacy of panitumumab (Pmab) when added to maintenance therapy with fluorouracil and folinic acid (FU/FA) in patients with RAS wild-type metastatic colorectal cancer. METHODS: Following first-line induction therapy with six cycles of FU/FA and oxaliplatin plus Pmab, responding patients (stable disease or partial or complete remission) were randomly assigned (1:1, open-label) to maintenance treatment with either FU/FA plus Pmab or FU/FA alone. The primary objective was to demonstrate superiority of progression-free survival (PFS, time from random assignment until progression or death) in favor of FU/FA plus Pmab with a hazard ratio (HR) of 0.75, a power of 80%, and a significance level of 10%. Secondary end points included overall survival, objective response rate of maintenance therapy, and toxicity. Survival end points were analyzed by the Kaplan-Meier method and compared by log-rank test and Cox regressions. Dichotomous variables were compared by Fisher's exact test; odds ratios were indicated when appropriate. The trial is registered with ClinicalTrials.gov (NCT01991873). RESULTS: Overall, 248 patients were randomly assigned and received maintenance therapy with either FU/FA plus Pmab (125 patients) or FU/FA alone (123 patients). At data cutoff, with 218 events (of 218 needed), PFS of maintenance therapy was significantly improved with FU/FA plus Pmab (8.8 months v 5.7 months; HR, 0.72; 80% CI, 0.60 to 0.85; P = .014). Overall survival (event rate 54%) numerically favored the FU/FA plus Pmab arm (28.7 months v 25.7 months; HR, 0.84; 95% CI, 0.60 to 1.18; P = .32). Objective response rates were 40.8% in patients receiving FU/FA plus Pmab versus 26.0% in patients receiving FU/FA alone (odds ratio, 1.96; 95% CI, 1.14 to 3.36; P = .02). The most frequent Common Terminology Criteria for Adverse Event grade ≥ 3 event during maintenance therapy was skin rash (7.2%). CONCLUSION: In RAS wild-type metastatic colorectal cancer, maintenance therapy with FU/FA plus Pmab induced a significantly superior PFS compared with FU/FA alone. If active maintenance therapy is aspired following induction therapy with FU/FA and oxaliplatin plus Pmab, FU/FA plus Pmab appears to be the most favorable option.

Relevance and clinical implications of tumor cell mobilization in the autologous transplant setting.

Autologous transplantation of peripheral blood (PB) hematopoietic stem cells (HSCs) is a widely used strategy for reconstitution of blood cells following high-dose chemotherapy for hematologic malignancies such as multiple myeloma (MM), non-Hodgkin lymphoma (NHL), and acute myeloid leukemia (AML), among others. Stem cells for transplantation are usually obtained from PB after treatment with chemotherapy with or without cytokine, usually granulocyte-colony stimulating factor (G-CSF), or after treatment with cytokine alone. The use of autologous peripheral blood stem cells (PBSCs) for transplantation is associated with the risk of contamination of the graft with tumor cells; whether this impacts response rates, progression-free survival (PFS), and overall survival (OS) is still debatable. This review summarizes the controversy surrounding tumor cell mobilization (TCM), the complexity of detection of minimal residual diseases, the available diagnostic tools, differences in TCM with available mobilization regimens, and the potential effect of TCM on clinical outcome. Collectively, these data suggest that new treatment paradigms to manage hematologic malignancies, such as MM, NHL, and AML, are needed and should focus on increasing the chemosensitivity of the tumor and eliminating residual disease.

Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma.

This phase 3, multicenter, randomized (1:1), double-blind, placebo-controlled study evaluated the safety and efficacy of plerixafor with granulocyte colony-stimulating factor (G-CSF) in mobilizing hematopoietic stem cells in patients with multiple myeloma. Patients received G-CSF (10 microg/kg) subcutaneously daily for up to 8 days. Beginning on day 4 and continuing daily for up to 4 days, patients received either plerixafor (240 microg/kg) or placebo subcutaneously. Starting on day 5, patients began daily apheresis for up to 4 days or until more than or equal to 6 x 10(6) CD34(+) cells/kg were collected. The primary endpoint was the percentage of patients who collected more than or equal to 6 x 10(6) CD34(+) cells/kg in less than or equal to 2 aphereses. A total of 106 of 148 (71.6%) patients in the plerixafor group and 53 of 154 (34.4%) patients in the placebo group met the primary endpoint (P < .001). A total of 54% of plerixafor-treated patients reached target after one apheresis, whereas 56% of the placebo-treated patients required 4 aphereses to reach target. The most common adverse events related to plerixafor were gastrointestinal disorders and injection site reactions. Plerixafor and G-CSF were well tolerated, and significantly more patients collected the optimal CD34(+) cell/kg target for transplantation earlier compared with G-CSF alone. This study is registered at www.clinicaltrials.gov as #NCT00103662.

Unrestricted somatic stem cells: interaction with CD34+ cells in vitro and in vivo, expression of homing genes and exclusion of tumorigenic potential.

BACKGROUND AIMS: Transplantation of allogeneic hematopoietic stem cells (HSC) within the framework of hematologic oncology or inherited diseases may be associated with complications such as engraftment failure and long-term pancytopenia. HSC engraftment can be improved, for example by co-transplantation with mesenchymal stem cells (MSC). Recently, a new multipotent MSC line from umbilical cord blood, unrestricted somatic stem cells (USSC), has been described. It was demonstrated that USSC significantly support proliferation of HSC in an in vitro feeder layer assay. METHODS: A NOD/SCID mouse model was used to assess the effect of USSC on co-transplanted CD34(+) cells and look for the fate of transplanted USSC. The migration potential of USSC was studied in a Boyden chamber migration assay and in vivo. Quantitative real-time polymerase chain reaction (qRT-PCR) for CXCR4, CD44, LFA1, CD62L, VLA4, RAC2, VLA5A and RAC1 were performed. NMR1 nu/nu mice were used for a tumorigenicity test. RESULTS: After 4 weeks, homing of human cells (CD45(+)) to the bone marrow of NOD/SCID mice was significantly increased in mice co-transplanted with CD34(+) cells and USSC (median 30.9%, range 7-50%) compared with the CD34(+) cell-only control group (median 5.9%, range 3-10%; P = 0.004). Homing of USSC could not be shown in the bone marrow. A cell-cell contact was not required for the graft enhancing effect of USSC. An in vivo tumorigenicity assay showed no tumorigenic potential of USSC. CONCLUSIONS: This pre-clinical study clearly shows that USSC have an enhancing effect on engraftment of human CD34(+) cells. USSC are a safe graft adjunct.

A Lentiviral CXCR4 overexpression and knockdown model in colorectal cancer cell lines reveals plerixafor-dependent suppression of SDF-1α-induced migration and invasion.

BACKGROUND: The development of distant metastasis is associated with poor outcome in patients with colorectal cancer (CRC). The stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) have pivotal roles in the chemotaxis of migrating tumor cells during metastasis. Thus, hampering the SDF-1/CXCR4 cross-talk is a promising strategy to suppress metastasis. METHODS: We investigated the invasive behavior of the lentivirally CXCR4 overexpressing CRC cell lines SW480, SW620 and RKO in chemotaxis and invasion assays toward an SDF-1α gradient. Low endogenous CXCR4 expression levels were determined by quantitative realtime polymerase chain reaction (PCR) and fluorescence-activated cell sorting (FACS) analyses. RESULTS: A lentiviral CXCR4 overexpression and knockdown model was established in these CRC cells. In transwell migration assays, CXCR4 overexpression favored chemotaxis and invasion of cells in all 3 lines depending on an SDF-1α gradient (p < 0.001 vs. untransduced cells). Functional CXCR4 knockdown using lentiviral short hairpin RNA (shRNA) vectors significantly decreased the migration behavior in CRC cell lines (p < 0.001), confirming a CXCR4-specific effect. Pharmacologic inhibition of the SDF-1α/CXCR4 interaction by the bicyclam Plerixafor(TM) at 100 µM significantly abrogated CXCR4-dependent migration and invasion through Matrigel(TM) (SW480, SW620, RKO; p < 0.05). CONCLUSION: Our results indicate that a CXCR4-antagonistic therapy might prevent tumor cell dissemination and metastasis in CRC patients, consequently improving survival.

Comparison of unmobilized and mobilized graft characteristics and the implications of cell subsets on autologous and allogeneic transplantation outcomes.

Autologous and allogeneic hematopoietic stem cell transplantation (HSCT) are considered the standard of care for many malignancies, including lymphoma, myeloma, and some leukemias. In many cases, mobilized peripheral blood has become the preferred source of hematopoietic stem cells. The efficacy of different mobilization regimens and transplantation outcomes based on cell doses has been well studied; however, the characteristics of the stem cell graft may be of equal importance with respect to patient outcomes following autologous or allogeneic HSCT. This review summarizes available preclinical and clinical data for bone marrow and mobilized peripheral blood HSCT characteristics, defined as the cell types found in the graft as well as their gene expression profiles. It also explores how graft characteristics can affect bone marrow homing, engraftment, immune reconstitution, and other posttransplantation outcomes in both the allogeneic and autologous HSCT settings.

Overnight transduction with foamyviral vectors restores the long-term repopulating activity of Fancc-/- stem cells.

Fanconi anemia (FA) is a complex genetic disorder characterized by congenital abnormalities, bone marrow failure, and myeloid malignancies. Identification of 13 FA genes has been instrumental to explore gene transfer technologies aimed at correction of autologous FA-deficient stem cells. To date, 3 human FA stem cell gene therapy trials with standard 4-day transduction protocols using gammaretroviral vectors failed to provide clinical benefit. In addition, 2- to 4 day ex vivo manipulation of bone marrow from mice containing a disruption of the homologue of human FANCC (Fancc) results in a time-dependent increase in apoptosis and a risk for malignant transformation of hematopoietic cells. Here, we show that a 14-hour transduction period allows a foamyviral vector construct expressing the human FANCC cDNA to efficiently transduce murine FA stem cells with 1 to 2 proviral integrations per genome. Functionally, the repopulating activity of Fancc(-/-) stem cells from reconstituted mice expressing the recombinant FANCC transgene was comparable with wild-type controls. Collectively, these data provide evidence that short-term transduction of c-kit(+) cells with a foamyviral vector is sufficient for functional correction of a stem cell phenotype in a murine FA model. These data could have implications for future gene therapy trials for FA patients.

Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34(+) peripheral blood progenitor cells.

BACKGROUND AND AIMS: Because of their pluripotency, human CD34(+) peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC. METHODS: A panel of pseudotyped AAV vectors (designated AAV2/x, containing the vector genome of serotype 2 and capsid of serotype x, AAV2/1-AAV2/6) was screened on primary human granulocyte-colony-stimulating factor (G-CSF)-mobilized CD34(+) PBPC to determine their gene transfer efficacy. Additionally, double-stranded self-complementary AAV (dsAAV) were used to determine possible second-strand synthesis limitations. RESULTS: AAV2/6 vectors proved to be the most efficient [12.8% (1.8-25.4%) transgene-expressing PBPC after a single transduction], being significantly more efficient (all P<0.005) than the other vectors [AAV2/2, 2.0% (0.2-7.3%); AAV2/1, 1.3% (0.1-2.9%); others, <; 1% transgene-expressing PBPC]. In addition, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using pseudotyped dsAAV vectors: for dsAAV2/2 [9.3% (8.3-20.3%); P<0.001] and dsAAV2/6 [37.7% (23.6-61.0%); P<0.001) significantly more PBPC expressed the transgene compared with their single-stranded counterparts; for dsAAV2/1, no significant increase could be observed. CONCLUSIONS: We have shown that clinically relevant transduction efficiency levels using AAV-based vectors in human CD34(+) PBPC are feasible, thereby offering an efficient alternative vector system for gene transfer into this important target cell population.

Normal-tissue radioprotection by overexpression of the copper-zinc and manganese superoxide dismutase genes.

BACKGROUND AND PURPOSE: Protection of normal tissue against radiation-induced damage may increase the therapeutic ratio of radiotherapy. A promising strategy for testing this approach is gene therapy-mediated overexpression of the copper-zinc (CuZnSOD) or manganese superoxide dismutase (MnSOD) using recombinant adeno-associated viral (rAAV2) vectors. The purpose of this study was to test the modulating effects of the SOD genes on human primary lung fibroblasts (HPLF) after irradiation. MATERIAL AND METHODS: HPLF were transduced with rAAV2 vectors containing cDNA for the CuZnSOD, MnSOD or a control gene. The cells were irradiated (1-6 Gy), and gene transfer efficiency, apoptosis, protein expression/activity, and radiosensitivity measured by the colony-forming assay determined. RESULTS: After transduction, 90.0% +/- 6.4% of the cells expressed the transgene. A significant fivefold overexpression of both SOD was confirmed by an SOD activity assay (control: 21.1 +/- 12.6, CuZnSOD: 95.1 +/- 17.1, MnSOD: 108.5 +/- 36.0 U SOD/mg protein) and immunohistochemistry. CuZnSOD and MnSOD overexpression resulted in a significant radioprotection of HPLF compared to controls (surviving fraction [SF] ratio SOD/control > 1): CuZnSOD: 1.18-fold (95% confidence interval [CI]: 1.06-1.32; p = 0.005), MnSOD: 1.23-fold (95% CI: 1.07-1.43; p = 0.01). CONCLUSION: Overexpression of CuZnSOD and MnSOD in HPLF mediated an increase in clonogenic survival after irradiation compared to controls. In previous works, a lack of radioprotection in SOD-overexpressing tumor cells was observed. Therefore, the present results suggest that rAAV2 vectors are promising tools for the delivery of radioprotective genes in normal tissue.

A combination of granulocyte-colony-stimulating factor (G-CSF) and plerixafor mobilizes more primitive peripheral blood progenitor cells than G-CSF alone: results of a European phase II study.

BACKGROUND AIMS: Previous studies in xenograft models have shown that human peripheral blood progenitor cells (PBPC) mobilized with the CXCR4 antagonist plerixafor (AMD3100) have a higher bone marrow (BM) reconstitution potential than granulocyte-colony-stimulating factor (G-CSF)-mobilized PBPC. METHODS: PBPC obtained during G-CSF-supported mobilization before and after a supplementary administration of AMD3100 from patients with multiple myeloma and non-Hodgkin's lymphoma (n=15; phase II study) were investigated for co-expression of primitive and lineage-associated markers, their proliferative activity in vitro and repopulation potential after clinical transplantation. RESULTS: A significant increase in primitive CD34+ CD38(-) cells was observed in intraindividual comparisons of all patients after administration of G-CSF+AMD3100 (peripheral blood: median 8-fold, range 2,4-fold - 39-fold) compared with G-CSF alone. Using a long-term culture-initiating cell assay, this increase was confirmed. After transplantation of G-CSF+AMD3100-mobilized PBPC, the time to leukocyte reconstitution > 1 x 10(3)/microL and platelet reconstitution > 2 x 10(4)/microL was 14 (10-19 days) and 13 days (10-15 days), respectively. A complete and stable hematologic reconstitution (platelets > 1.5 x 10(5)/microL) was observed in 91% of all patients within 35 days. CONCLUSIONS: An additional application of AMD3100 to a standard G-CSF mobilization regimen leads to a significant increase in primitive PBPC with high repopulation capacity.

Generation of efficient human blood progenitor-targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells.

OBJECTIVE: Currently standard recombinant adeno-associated virus serotype 2(rAAV2)-based vectors lack the efficiency for gene transfer into primary human CD34(+) peripheral blood progenitor cells (PBPC). MATERIALS AND METHODS: An advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34(+) PBPC cells. RESULTS: On a panel of leukemia cell lines (CML/AML), significantly higher gene transfer efficiency of the rAAV capsid mutants (up to 100% gene transfer) was observed compared to standard rAAV2 vectors. A higher transduction efficiency in the imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S and a pronounced difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S were particularly striking. On solid tumor cell lines, on the other hand, rAAV2 was more efficient than the capsid mutants, suggesting an increased specificity of our capsid mutants for hematopoietic progenitor cells. On primary human CD34(+) PBPC significantly higher (up to eightfold; 16% green fluorescent protein-positive) gene transfer could be obtained with the newly generated vectors compared to standard rAAV2 vectors. CONCLUSION: These novel vectors may enable efficient gene transfer using rAAV-based vectors into primary human blood progenitor cells for a future clinical application.