Perioperative chemotherapy with fluorouracil plus leucovorin, oxaliplatin, and docetaxel versus fluorouracil or capecitabine plus cisplatin and epirubicin for locally advanced, resectable gastric or gastro-oesophageal junction adenocarcinoma (FLOT4): a randomised, phase 2/3 trial.

BACKGROUND: Docetaxel-based chemotherapy is effective in metastatic gastric and gastro-oesophageal junction adenocarcinoma. This study reports on the safety and efficacy of the docetaxel-based triplet FLOT (fluorouracil plus leucovorin, oxaliplatin and docetaxel) as a perioperative therapy for patients with locally advanced, resectable tumours. METHODS: In this controlled, open-label, phase 2/3 trial, we randomly assigned 716 patients with histologically-confirmed advanced clinical stage cT2 or higher or nodal positive stage (cN+), or both, resectable tumours, with no evidence of distant metastases, via central interactive web-based-response system, to receive either three pre-operative and three postoperative 3-week cycles of 50 mg/m2 epirubicin and 60 mg/m2 cisplatin on day 1 plus either 200 mg/m2 fluorouracil as continuous intravenous infusion or 1250 mg/m2 capecitabine orally on days 1 to 21 (ECF/ECX; control group) or four preoperative and four postoperative 2-week cycles of 50 mg/m2 docetaxel, 85 mg/m2 oxaliplatin, 200 mg/m2 leucovorin and 2600 mg/m2 fluorouracil as 24-h infusion on day 1 (FLOT; experimental group). The primary outcome of the trial was overall survival (superiority) analysed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01216644. FINDINGS: Between Aug 8, 2010, and Feb 10, 2015, 716 patients were randomly assigned to treatment in 38 German hospitals or with practice-based oncologists. 360 patients were assigned to ECF/ECX and 356 patients to FLOT. Overall survival was increased in the FLOT group compared with the ECF/ECX group (hazard ratio [HR] 0·77; 95% confidence interval [CI; 0.63 to 0·94]; median overall survival, 50 months [38·33 to not reached] vs 35 months [27·35 to 46·26]). The number of patients with related serious adverse events (including those occurring during hospital stay for surgery) was similar in the two groups (96 [27%] in the ECF/ECX group vs 97 [27%] in the FLOT group), as was the number of toxic deaths (two [<1%] in both groups). Hospitalisation for toxicity occurred in 94 patients (26%) in the ECF/ECX group and 89 patients (25%) in the FLOT group. INTERPRETATION: In locally advanced, resectable gastric or gastro-oesophageal junction adenocarcinoma, perioperative FLOT improved overall survival compared with perioperative ECF/ECX. FUNDING: The German Cancer Aid (Deutsche Krebshilfe), Sanofi-Aventis, Chugai, and Stiftung Leben mit Krebs Foundation.

High microsatellite instability (MSI-H) is associated with distinct clinical and molecular characteristics and an improved survival in early Colon cancer (CC); real world data from the AIO molecular registry Colopredict Plus.

Colorectal cancer is one of the leading malignancies and still accounts for almost 25 000 deaths in Germany each year. Although there is accumulating data on the molecular basis, treatment and clinical outcome of patients within clinical trials evidence from the real-world setting is mostly lacking. We started the molecular registry trial Colopredict Plus in 2013 to collect clinical and molecular data from a real-world cohort of patients with early colon cancer stage II and III in 70 German colon cancer centers focusing on the prognostic impact of high microsatellite instability. In this interim report, we characterize a clinical cohort of 2615 patients, of whom 1787 tissue probes were analyzed. Microsatellite status was assessed using immunhistochemistry and fragment length analysis, with a concordance of 91.4 %. These established histopathological methods are sensitive and cost-effective. The median age was 72 years, significantly higher compared to clinical trial populations, with a median Charlson Comorbidity Index of 3. The stage-dependent incidence of microsatellite instability was 23.7 % and was associated with female gender, BRAF-mutation, UICC stage II and localization in the right colon. Survival calculated in disease free, relapse free and overall survival significantly differed between MSI-H and MSS, in favor of MSI-H patients. Multivariate age-adjusted analyses of relapse-free survival, disease-free survival, and overall survival highlighted microsatellite instability as a robust and positive prognostic marker for early colon cancer independent of age.

Administration of bortezomib before and after autologous stem cell transplantation improves outcome in multiple myeloma patients with deletion 17p.

In patients with multiple myeloma (MM), risk stratification by chromosomal abnormalities may enable a more rational selection of therapeutic approaches. In the present study, we analyzed the prognostic value of 12 chromosomal abnormalities in a series of 354 MM patients treated within the HOVON-65/GMMG-HD4 trial. Because of the 2-arm design of the study, we were able to analyze the effect of a bortezomib-based treatment before and after autologous stem cell transplantation (arm B) compared with standard treatment without bortezomib (arm A). For allanalyzed chromosomal aberrations, progression-free survival (PFS) and overall survival (OS) were at least equal or superior in the bortezomib arm compared with the standard arm. Strikingly, patients with del(17p13) benefited the most from the bortezomib-containing treatment: the median PFS in arm A was 12.0 months and in arm B it was 26.2 months (P = .024); the 3 year-OS for arm A was 17% and for arm B it was 69% (P = .028). After multivariate analysis, del(17p13) was an independent predictor for PFS (P < .0001) and OS (P < .0001) in arm A, whereas no statistically significant effect on PFS (P = .28) or OS (P = .12) was seen in arm B. In conclusion, the adverse impact of del(17p13) on PFS and OS could be significantly reduced by bortezomib-based treatment, suggesting that long-term administration of bortezomib should be recommended for patients carrying del(17p13).

Fast and label-free automated detection of microsatellite status in early colon cancer using artificial intelligence integrated infrared imaging.

PURPOSE: Microsatellite instability (MSI) due to mismatch repair (MMR) defects accounts for 15-20% of colon cancers (CC). MSI testing is currently standard of care in CC with immunohistochemistry of the four MMR proteins representing the gold standard. Instead, label-free quantum cascade laser (QCL) based infrared (IR) imaging combined with artificial intelligence (AI) may classify MSI/microsatellite stability (MSS) in unstained tissue sections user-independently and tissue preserving. METHODS: Paraffin-embedded unstained tissue sections of early CC from patients participating in the multicentre AIO ColoPredict Plus (CPP) 2.0 registry were analysed after dividing into three groups (training, test, and validation). IR images of tissue sections using QCL-IR microscopes were classified by AI (convolutional neural networks [CNN]) using a two-step approach. The first CNN (modified U-Net) detected areas of cancer while the second CNN (VGG-Net) classified MSI/MSS. End-points were area under receiver operating characteristic (AUROC) and area under precision recall curve (AUPRC). RESULTS: The cancer detection in the first step was based on 629 patients (train n = 273, test n = 138, and validation n = 218). Resulting classification AUROC was 1.0 for the validation dataset. The second step classifying MSI/MSS was performed on 547 patients (train n = 331, test n = 69, and validation n = 147) reaching AUROC and AUPRC of 0.9 and 0.74, respectively, for the validation cohort. CONCLUSION: Our novel label-free digital pathology approach accurately and rapidly classifies MSI vs. MSS. The tissue sections analysed were not processed leaving the sample unmodified for subsequent analyses. Our approach demonstrates an AI-based decision support tool potentially driving improved patient stratification and precision oncology in the future.

Lasting response by vertical inhibition with cetuximab and trametinib in KRAS-mutated colorectal cancer patient-derived xenografts.

Although approximately half of all metastatic colorectal cancers (mCRCs) harbour mutations in KRAS or NRAS, hardly any progress has been made regarding targeted treatment for this group over the last few years. Here, we investigated the efficacy of vertical inhibition of the RAS-pathway by targeting epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase kinase (MEK) in patient-derived xenograft (PDX) tumours with primary KRAS mutation. In total, 19 different PDX models comprising 127 tumours were tested. Responses were evaluated according to baseline tumour volume changes and graded as partial response (PR; ≤ - 30%), stable disease (SD; between -30% and +20%) or progressive disease (PD; ≥ + 20%). Vertical inhibition with trametinib and cetuximab induced SD or PR in 74% of analysed models, compared to 24% by monotherapy with trametinib. In cases of PR by vertical inhibition (47%), responses were lasting (as long as day 137), with a low incidence of secondary resistance (SR). Molecular analyses revealed that primary and SR was driven by transcriptional reprogramming activating the RAS pathway in a substantial fraction of tumours. Together, these preclinical data strongly support the translation of this combination therapy into clinical trials for CRC patients.

Coinhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription.

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.

Causes of mortality in elderly UICC stage III colon cancer (CC) patients--Tumor-related death and competing risks from the German AIO colorectal study group Colopredict Plus (CPP) registry.

BACKGROUND: Colon cancer (CC) is a disease of elderly patients (pts.) with a median age of 73 years (yrs.). Lack of data about the effects of adjuvant chemotherapy (ACT) is caused by underrepresentation of this clinically relevant cohort in interventional trials. We analyzed real-world data from the German CPP registry with regard to a possible benefit of ACT in elderly (70+ yrs.) versus younger pts. (50 to <70 yrs.) taking cause-specific deaths into account. METHODS: We analyzed the effect of age and ACT on overall survival (OS) and cause-specific death of stage III pts. using Cox regression. RESULTS: In total, 1558 pts. were analyzed and follow-up was 24.6 months. 62.6% of the elderly received ACT whereas 91.1% of younger pts. (p < 0.001). Oxaliplatin combinations were significantly less often given to older than younger pts. (38.8% vs. 88.9%; p < 0.001). Mean Charlson comorbidity score was significantly lower in pts. that received ACT (0.61) than in those without ACT (1.16; p < 0.001). ACT was an independent positive prognostic factor for cancer-related death in elderly pts. even in pts. 75+ yrs. No significant difference in the effect of ACT could be observed between age groups (interaction: cancer-specific death HR = 1.7948, p = 0.1079; death of other cause HR = 0.7384, p = 0.6705). CONCLUSION: ACT was an independent positive prognostic factor for OS. There may be a cohort of elderly with less co-morbidities who benefit from ACT.

FLOT Versus FLOT/Trastuzumab/Pertuzumab Perioperative Therapy of Human Epidermal Growth Factor Receptor 2-Positive Resectable Esophagogastric Adenocarcinoma: A Randomized Phase II Trial of the AIO EGA Study Group.

PURPOSE: High pathologic complete response (pCR) rates and comparably good survival data were seen in a phase II trial combining perioperative fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT) chemotherapy with trastuzumab for resectable, esophagogastric adenocarcinoma (EGA). The current trial evaluates the addition of trastuzumab and pertuzumab to FLOT as perioperative treatment for human epidermal growth factor receptor 2-positive resectable EGA. METHODS: In this multicenter, randomized phase II/III trial, patients with human epidermal growth factor receptor 2-positive, resectable EGA (≥ clinical tumor 2 or clinical nodal-positive) were assigned to four pre- and postoperative cycles of either FLOT alone (arm A) or combined with trastuzumab and pertuzumab, followed by nine cycles of trastuzumab/pertuzumab (arm B). The primary end point for the phase II part was the rate of pCR. RESULTS: The trial was closed prematurely, without transition into phase III, after results of the JACOB trial were reported. Eighty-one patients were randomly assigned (A: 41/B: 40) during the phase II part. The pCR rate was significantly improved with the trastuzumab/pertuzumab treatment (A: 12%/B: 35%; P = .02). Similarly, the rate of pathologic lymph node negativity was higher with trastuzumab/pertuzumab (A: 39%/B: 68%), whereas the R0 resection rate (A: 90%/B: 93%) and surgical morbidity (A: 43%/B: 44%) were comparable. Moreover, the inhouse mortality was equal in both arms (overall 2.5%). The median disease-free survival was 26 months in arm A and not yet reached in arm B (hazard ratio, 0.58; P = .14). After a median follow-up of 22 months, the median overall survival was not yet reached (hazard ratio, 0.56; P = .24). Disease-free survival and overall survival rates at 24 months were 54% (95% CI, 38 to 71) and 77% (95% CI, 63 to 90) in arm A and 70% (95% CI, 55 to 85) and 84% (95% CI, 72 to 96) in arm B, respectively. More ≥ grade 3 adverse events were reported with trastuzumab/pertuzumab, especially diarrhea (A: 5%/B: 41%) and leukopenia (A: 13%/B: 23%). CONCLUSION: The addition of trastuzumab/pertuzumab to perioperative FLOT significantly improved pCR and nodal negativity rates at the price of higher rates of diarrhea and leukopenia.

A randomized, phase III trial of capecitabine plus bevacizumab (Cape-Bev) versus capecitabine plus irinotecan plus bevacizumab (CAPIRI-Bev) in first-line treatment of metastatic colorectal cancer: the AIO KRK 0110 trial/ML22011 trial.

BACKGROUND: Several randomized trials have indicated that combination chemotherapy applied in metastatic colorectal cancer (mCRC) does not significantly improve overall survival when compared to the sequential use of cytotoxic agents (CAIRO, MRC Focus, FFCD 2000-05). The present study investigates the question whether this statement holds true also for bevacizumab-based first-line treatment including escalation- and de-escalation strategies. METHODS/DESIGN: The AIO KRK 0110/ML22011 trial is a two-arm, multicenter, open-label randomized phase III trial comparing the efficacy and safety of capecitabine plus bevacizumab (Cape-Bev) versus capecitabine plus irinotecan plus bevacizumab (CAPIRI-Bev) in the first-line treatment of metastatic colorectal cancer. Patients with unresectable metastatic colorectal cancer, Eastern Cooperative Oncology Group (ECOG) performance status 0-1, will be assigned in a 1:1 ratio to receive either capecitabine 1250 mg/m(2) bid for 14d (d1-14) plus bevacizumab 7.5 mg/kg (d1) q3w (Arm A) or capecitabine 800 mg/m(2) BID for 14d (d1-14), irinotecan 200 mg/m(2) (d1) and bevacizumab 7.5 mg/kg (d1) q3w (Arm B). Patients included into this trial are required to consent to the analysis of tumour tissue and blood for translational investigations. In Arm A, treatment escalation from Cape-Bev to CAPIRI-Bev is recommended in case of progressive disease (PD). In Arm B, de-escalation from CAPIRI-Bev to Cape-Bev is possible after 6 months of treatment or in case of irinotecan-associated toxicity. Re-escalation to CAPIRI-Bev after PD is possible. The primary endpoint is time to failure of strategy (TFS). Secondary endpoints are overall response rate (ORR), overall survival, progression-free survival, safety and quality of life. CONCLUSION: The AIO KRK 0110 trial is designed for patients with disseminated, but asymptomatic mCRC who are not potential candidates for surgical resection of metastasis. Two bevacizumab-based strategies are compared: one starting as single-agent chemotherapy (Cape-Bev) allowing escalation to CAPIRI-Bev and another starting with combination chemotherapy (CAPIRI-Bev) and allowing de-escalation to Cape-Bev and subsequent re-escalation if necessary. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01249638 EudraCT-No.: 2009-013099-38.

Secondary resistance to anti-EGFR therapy by transcriptional reprogramming in patient-derived colorectal cancer models.

BACKGROUND: The development of secondary resistance (SR) in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (anti-EGFR) antibodies is not fully understood at the molecular level. Here we tested in vivo selection of anti-EGFR SR tumors in CRC patient-derived xenograft (PDX) models as a strategy for a molecular dissection of SR mechanisms. METHODS: We analyzed 21 KRAS, NRAS, BRAF, and PI3K wildtype CRC patient-derived xenograft (PDX) models for their anti-EGFR sensitivity. Furthermore, 31 anti-EGFR SR tumors were generated via chronic in vivo treatment with cetuximab. A multi-omics approach was employed to address molecular primary and secondary resistance mechanisms. Gene set enrichment analyses were used to uncover SR pathways. Targeted therapy of SR PDX models was applied to validate selected SR pathways. RESULTS: In vivo anti-EGFR SR could be established with high efficiency. Chronic anti-EGFR treatment of CRC PDX tumors induced parallel evolution of multiple resistant lesions with independent molecular SR mechanisms. Mutations in driver genes explained SR development in a subgroup of CRC PDX models, only. Transcriptional reprogramming inducing anti-EGFR SR was discovered as a common mechanism in CRC PDX models frequently leading to RAS signaling pathway activation. We identified cAMP and STAT3 signaling activation, as well as paracrine and autocrine signaling via growth factors as novel anti-EGFR secondary resistance mechanisms. Secondary resistant xenograft tumors could successfully be treated by addressing identified transcriptional changes by tailored targeted therapies. CONCLUSIONS: Our study demonstrates that SR PDX tumors provide a unique platform to study molecular SR mechanisms and allow testing of multiple treatments for efficient targeting of SR mechanisms, not possible in the patient. Importantly, it suggests that the development of anti-EGFR tolerant cells via transcriptional reprogramming as a cause of anti-EGFR SR in CRC is likely more prevalent than previously anticipated. It emphasizes the need for analyses of SR tumor tissues at a multi-omics level for a comprehensive molecular understanding of anti-EGFR SR in CRC.

Preclinical Efficacy of Covalent-Allosteric AKT Inhibitor Borussertib in Combination with Trametinib in KRAS-Mutant Pancreatic and Colorectal Cancer.

Aberrations within the PI3K/AKT signaling axis are frequently observed in numerous cancer types, highlighting the relevance of these pathways in cancer physiology and pathology. However, therapeutic interventions employing AKT inhibitors often suffer from limitations associated with target selectivity, efficacy, or dose-limiting effects. Here we present the first crystal structure of autoinhibited AKT1 in complex with the covalent-allosteric inhibitor borussertib, providing critical insights into the structural basis of AKT1 inhibition by this unique class of compounds. Comprehensive biological and preclinical evaluation of borussertib in cancer-related model systems demonstrated a strong antiproliferative activity in cancer cell lines harboring genetic alterations within the PTEN, PI3K, and RAS signaling pathways. Furthermore, borussertib displayed antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models of mutant KRAS pancreatic and colon cancer. SIGNIFICANCE: Borussertib, a first-in-class covalent-allosteric AKT inhibitor, displays antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models and provides a starting point for further pharmacokinetic/dynamic optimization.

Induction of interleukin-6 by hepatitis C virus core protein in hepatitis C-associated mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma.

PURPOSE: Chronic hepatitis C carries the risk to develop mixed cryoglobulinemia (MC) and B-cell non-Hodgkin's lymphoma (B-NHL), possibly because viral antigens stimulate the host's inflammatory response via extracellular pattern recognition receptors (PRR). To clarify this issue, we studied whether recognition of hepatitis C virus (HCV) proteins by PRR is involved in the pathogenesis of HCV-associated MC or B-NHL. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells of patients with HCV-associated B-NHL (n = 12), MC (n = 14), uncomplicated hepatitis C (n = 12), and healthy volunteers (n = 12) were incubated with the recombinant HCV proteins E2, core, and NS3 to study induction of cytokine production, stimulation of B-cell proliferation, and immunoglobulin secretion. In addition, serum levels of interleukin-6 (IL-6) were measured by ELISA. RESULTS: HCV core was the only studied protein, which induced production of IL-6 and IL-8 in CD14(+) cells. IL-6 induction was mediated via Toll-like receptor 2 (TLR2) and lead to increased B-cell proliferation in vitro. TLR2 expression on monocytes and IL-6 serum concentrations were increased in all groups of HCV-infected patients compared with healthy controls and were highest in MC (P < 0.05). CONCLUSIONS: Increased secretion of IL-6 via stimulation of TLR2 by HCV core protein may play a role in the pathogenesis of hepatitis C-associated MC and B-NHL.

Blood-based detection of RAS mutations to guide anti-EGFR therapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing.

An accurate blood-based RAS mutation assay to determine eligibility of metastatic colorectal cancer (mCRC) patients for anti-EGFR therapy would benefit clinical practice by better informing decisions to administer treatment independent of tissue availability. The objective of this study was to determine the level of concordance between plasma and tissue RAS mutation status in patients with mCRC to gauge whether blood-based RAS mutation testing is a viable alternative to standard-of-care RAS tumor testing. RAS testing was performed on plasma samples from newly diagnosed metastatic patients, or from recurrent mCRC patients using the highly sensitive digital PCR technology, BEAMing (beads, emulsions, amplification, and magnetics), and compared with DNA sequencing data of respective FFPE (formalin-fixed paraffin-embedded) tumor samples. Discordant tissue RAS results were re-examined by BEAMing, if possible. The prevalence of RAS mutations detected in plasma (51%) vs. tumor (53%) was similar, in accord with the known prevalence of RAS mutations observed in mCRC patient populations. The positive agreement between plasma and tumor RAS results was 90.4% (47/52), the negative agreement was 93.5% (43/46), and the overall agreement (concordance) was 91.8% (90/98). The high concordance of plasma and tissue results demonstrates that blood-based RAS mutation testing is a viable alternative to tissue-based RAS testing.

Recombinant adeno-associated and adenoviral vectors for the transduction of pancreatic and colon carcinoma.

BACKGROUND: Recombinant adeno-associated viral vectors serotype 2 (rAAV2) can transduce several tissues with high efficiency. MATERIALS AND METHODS: The transduction efficiency of rAAV2 in pancreatic and colon cancer was compared to that of recombinant adenovirus (rAd) in vitro and in vivo using green fluorescent protein (GFP). RESULTS: With the exception of SU.86.86, the percentage of GFP-positive cells was below 10% at a multiplicity of infection (MOI) of 100 for rAAV2. At the same MOI, almost 100% of cells expressed GFP when rAd was used. However, the transduction efficiency for rAA V2 was comparable to that of rAd when coinfected with wt adenovirus, leading to a dramatic increase in the amount of double-stranded rAAV2 DNA. Similar results were obtained in vivo. While widespread GFP expression was readily detected in all xenografts injected with rAd, only one section of all tumors injected with rAAV2 contained GFP-positive cells. CONCLUSION: The results of this study indicate that rAAV2 might be useful for an ex vivo approach in cancer gene therapy, but it does not seem to be feasible for the in vivo treatment of malignant tumors.

Combination of bortezomib, thalidomide, and dexamethasone in the treatment of relapsed, refractory IgD multiple myeloma.

The use of the proteasome inhibitor bortezomib has been recently introduced into the treatment of relapsed, refractory multiple myeloma (MM). We here demonstrate a case of a patient with IgD MM who was successfully treated with a combination of bortezomib, thalidomide, and dexamethasone. Relapse of the disease occurred following tandem autologous transplantation and was refractory to a salvage therapy consisting of thalidomide, cyclophosphamide, etoposide, and dexamethasone. While the administration of thalidomide was continued, the addition of bortezomib and dexamethasone led to a complete remission after 2 cycles of therapy. The feasibility of this treatment is supported by a recently reported phase I/II trial that used a lower dose of thalidomide in combination with an equal dose of bortezomib. This is the first report of a patient with IgD MM treated accordingly, suggesting that this entity is highly sensitive to the novel therapy.

Adeno-associated virus-mediated gene transfer of endostatin inhibits angiogenesis and tumor growth in vivo.

A variety of approaches has demonstrated that interfering with tumor-induced angiogenesis may be an effective strategy in cancer therapy. However, it is likely that to be most effective such strategies will require extended suppression of the angiogenic process. Gene therapy offers a possible approach to achieve sustained release of a therapeutically potent transferred gene product. In the present study the angiogenesis inhibitor endostatin was expressed through a recombinant adeno-associated viral (rAAV) vector and shown to be biologically active in vitro and in vivo. Intramuscular injection of rAAV-HuEndo (1 x 10(9) i.u.) led to a sustained serum endostatin level of approximately 35-40 ng/mL. This endostatin level was sufficient to inhibit tumor cell-induced angiogenesis and to suppress both the initiation and subsequent growth of a human colorectal cancer model.

Gene therapy delivery of endostatin enhances the treatment efficacy of radiation.

BACKGROUND AND PURPOSE: To evaluate whether sustained expression of mouse endostatin by adeno-associated virus (AAV)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation. MATERIALS AND METHODS: Mouse endostatin was cloned into recombinant AAV (rAAV) under the control of CMV beta-actin promoter. Recombinant mouse endostatin expressed via AAV gene transfer was tested for biological activity in endothelial cells. The impact of elevated serum levels of endostatin on tumor-induced angiogenesis was evaluated using an in vivo angiogenesis assay. The anti-tumor efficacy of combining rAAV-mediated endostatin delivery with radiation was evaluated in a human colorectal tumor model (HT29). RESULTS: Recombinant mouse endostatin expressed through an AAV vector (rAAV-mEndo) inhibited endothelial cell proliferation (by 40-45%) and migration (by 22-33%). Intramuscular injection of rAAV-mEndo (1x10(9) i.u.) led to a sustained serum endostatin level of approximately 500 ng/ml. Compared to control animals this endostatin level was sufficient to inhibit tumor cell-induced vessel formation (37 vs. 28.5, P<0.05) and delay the growth of HT29 xenografts (time from 200 to 1,000 mm(3), 21 vs. 34.5 days, P<0.05). When combined with ionizing radiation, elevated serum endostatin levels significantly enhanced the time for tumors to grow from 200 to 1,000 mm(3) (radiation, 34 days; endostatin plus radiation, 50 days, P<0.05). CONCLUSION: The delivery of endostatin via rAAV vectors may provide an effective means of enhancing the anti-tumor efficacy of radiation therapy.

Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus.

BACKGROUND: Silencing of the viral CMV immediate early enhancer promoter can be a problem in certain cell types when engineering stable cell lines. MATERIALS AND METHODS: We compared the efficacy of the CMV promoter to the promoter of the elongation factor-1 alpha (EF-1 alpha) for the generation of stable colon carcinoma cell lines (HT-29). Green fluorescent protein (GFP) expression cassettes were delivered by recombinant adeno-associated virus (AAV) which is known for its ability to stably transduce cells. Stable cell lines were characterized in vitro by FACS and in vivo after HT-29 clones were grown as xenografts in nude mice. RESULTS: Stable HT-29 clones with > 97% of all cells homogeneously expressing GFP were generated with the EF-1 alpha promoter. In contrast in clones carrying the CMV promoter, only up to 60% of the cells were GFP-positive with expression levels varying widely between cells. Superinfection with wild-type adenovirus induced GFP expression in more than 90% of the cells indicating that the CMV promoter was silenced. In vivo the tumors carrying the EF-1 alpha promoter were homogeneously GFP-positive, whereas the CMV promoter gave rise to a scattered pattern of GFP expression. CONCLUSION: This study underlines the importance of the promoter for the generation of stable cell lines. In addition it demonstrates that recombinant AAV can effectively be used as a gene delivery system for this purpose.

Efficacy of recombinant adeno-associated viral vectors serotypes 1, 2, and 5 for the transduction of pancreatic and colon carcinoma cells.

BACKGROUND: The development of efficient and specific vector systems remains a central issue in gene therapy. Several different adeno-associated virus (AAV) serotypes have so far been characterized so far which show different tissue tropisms. MATERIALS AND METHODS: The vectors used here contained AAV2 transgene cassette containing green fluorescent protein (GFP) in AAV1, AAV2, or AAV5 capsids, producing the recombinant pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5. The transduction efficiency of the different pseudotyped AAV vectors was tested in vitro in pancreatic and colon cancer cells lines (HT-29, BXPC3, and Hs766T). RESULTS: For all three serotypes, the percentage of GFP-positive cells was below 10% at multiplicities of infection (MOI) 100 rAAV vectors when used alone for infection. However, transduction efficiency for rAAV vectors increased dramatically when the cells were co-infected with wild-type adenovirus (wtAd). The percentage of GFP-positive cells ranged from 19.8-65.3% for AAV2/1 and 16.9-70.2% for AAV2/5, respectively. It was highest for rAAV2/2, at 40.9-88.4%. Variation between the cell lines was observed, with BXPC3 scoring the highest transduction rates and HT-29 the lowest. CONCLUSION: This study indicates that vectors based on distinct AAV serotypes 1, 2, and 5 all transduce pancreatic and colon cell lines poorly when used alone. Co-infection with wtAd increase transduction rates dramatically indicating that slow second-strand synthesis is a reason for the poor transduction efficiency. Due to the poor transduction rates, none of the rAAV serotypes tested here seem to be feasible for the treatment of malignant tumors.