Publications by authors named "Gunnar Kvalheim"

80 Publications

"Built-in" PD-1 blocker to rescue NK-92 activity from PD-L1-mediated tumor escape mechanisms.

FASEB J 2021 09;35(9):e21750

Department of Cellular Therapy, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway.

Success of adoptive cell therapy mainly depends on the ability of immune cells to persist and function optimally in the immunosuppressive tumor microenvironment. Although present at the cancer site, immune cells become exhausted and/or inhibited, due to the presence of inhibitory receptors such as PD-L1 on malignant cells. Novel genetic strategies to manipulate the PD1/PD-L1 axis comprise (i) PD-1 reversion where the receptor intracellular domain is replaced with an activating unit, (ii) the use of anti-PD-L1 CAR or (iii) the disruption of the PD-1 gene. We here present an alternative strategy to equip therapeutic cells with a truncated PD-1 (tPD-1) to abrogate PD-1/PD-L1 inhibition. We show that engagement of tPD-1 with PD-L1-positive tumor unleashes NK-92 activity in vitro. Furthermore, this binding was sufficiently strong to induce killing of targets otherwise not recognized by NK-92, thus increasing the range of targets. In vivo treatment with NK-92 tPD-1 cells led to reduced tumor growth and improved survival. Importantly, tPD-1 did not interfere with tumor recognition in PD-L1 negative conditions. Thus, tPD-1 represents a straightforward method for improving antitumor immunity and revealing new targets through PD-L1 positivity.
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http://dx.doi.org/10.1096/fj.202100025RDOI Listing
September 2021

Targeting KRAS mutations with HLA class II-restricted TCRs for the treatment of solid tumors.

Oncoimmunology 2021 17;10(1):1936757. Epub 2021 Jun 17.

Translational Research Unit, Department of Cellular Therapy, Oslo University Hospital, Oslo, Norway.

T-cell receptor (TCR) redirected T cells are considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers and are thus regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs as well as target-specific functionality when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate Human leukocyte antigen (HLA), suggesting that, here, the point mutations were less important for the HLA binding and TCR recognition, whereas others were single-mutation restricted. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR-based cancer immunotherapy.
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http://dx.doi.org/10.1080/2162402X.2021.1936757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216182PMC
August 2021

Combinatorial CAR design improves target restriction.

J Biol Chem 2021 Jan-Jun;296:100116. Epub 2020 Dec 3.

Department of Cellular Therapy, Department of Oncology, Oslo University Hospital, Oslo, Norway. Electronic address:

CAR T cells targeting the B lymphocyte antigen CD19 have led to remarkable clinical results in B cell leukemia and lymphoma but eliminate all B lineage cells, leading to increased susceptibility to severe infections. As malignant B cells will express either immunoglobulin (Ig) light chain κ or λ, we designed a second-generation CAR targeting Igκ, IGK CAR. This construct demonstrated high target specificity but displayed reduced efficacy in the presence of serum IgG. Since CD19 CAR is insensitive to serum IgG, we designed various combinatorial CAR constructs in order to maintain the CD19 CAR T cell efficacy, but with IGK CAR target selectivity. The Kz-19BB design, combining CD19 CAR containing a 4-1BB costimulatory domain with an IGK CAR containing a CD3zeta stimulatory domain, maintained the target specificity of IgK CAR and was resistant to the presence of soluble IgG. Our results demonstrate that a combinatorial CAR approach can improve target selectivity and efficacy.
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http://dx.doi.org/10.1074/jbc.RA120.016234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948400PMC
August 2021

Targeting Telomerase with an HLA Class II-Restricted TCR for Cancer Immunotherapy.

Mol Ther 2021 03 17;29(3):1199-1213. Epub 2020 Nov 17.

Department of Cellular Therapy, Oslo University Hospital-The Norwegian Radium Hospital, 0379 Oslo, Norway. Electronic address:

T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4 and CD8 T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.
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http://dx.doi.org/10.1016/j.ymthe.2020.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934585PMC
March 2021

Toll-like receptor 7/8-matured RNA-transduced dendritic cells as post-remission therapy in acute myeloid leukaemia: results of a phase I trial.

Clin Transl Immunology 2020 3;9(3):e1117. Epub 2020 Mar 3.

Department of Medicine III University Hospital, LMU Munich Munich Germany.

Objectives: Innovative post-remission therapies are needed to eliminate residual AML cells. DC vaccination is a promising strategy to induce anti-leukaemic immune responses.

Methods: We conducted a first-in-human phase I study using TLR7/8-matured DCs transfected with RNA encoding the two AML-associated antigens WT1 and PRAME as well as CMVpp65. AML patients in CR at high risk of relapse were vaccinated 10× over 26 weeks.

Results: Despite heavy pretreatment, DCs of sufficient number and quality were generated from a single leukapheresis in 11/12 cases, and 10 patients were vaccinated. Administration was safe and resulted in local inflammatory responses with dense T-cell infiltration. In peripheral blood, increased antigen-specific CD8 T cells were seen for WT1 (2/10), PRAME (4/10) and CMVpp65 (9/10). For CMVpp65, increased CD4 T cells were detected in 4/7 patients, and an antibody response was induced in 3/7 initially seronegative patients. Median OS was not reached after 1057 days; median RFS was 1084 days. A positive correlation was observed between clinical benefit and younger age as well as mounting of antigen-specific immune responses.

Conclusions: Administration of TLR7/8-matured DCs to AML patients in CR at high risk of relapse was feasible and safe and resulted in induction of antigen-specific immune responses. Clinical benefit appeared to occur more likely in patients <65 and in patients mounting an immune response. Our observations need to be validated in a larger patient cohort. We hypothesise that TLR7/8 DC vaccination strategies should be combined with hypomethylating agents or checkpoint inhibition to augment immune responses.

Trial Registration: The study was registered at https://clinicaltrials.gov on 17 October 2012 (NCT01734304) and at https://www.clinicaltrialsregister.eu (EudraCT-Number 2010-022446-24) on 10 October 2013.
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http://dx.doi.org/10.1002/cti2.1117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053229PMC
March 2020

Immune stimulatory effect of anti-EpCAM immunotoxin - improved overall survival of metastatic colorectal cancer patients.

Acta Oncol 2020 Apr 26;59(4):404-409. Epub 2019 Dec 26.

Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway.

In a recent phase I trial in a heterogeneous group of carcinoma patients with advanced disease, we did not observe objective responses by CT at 8 weeks in patients treated with either the anti-EpCAM immunotoxin MOC31PE alone or administered in combination with the immunosuppressor cyclosporin (CsA). We have now assessed overall survival (OS) data for the two groups to reveal potential differences, and to elucidate putative underlying mechanisms. The OS time of MOC31PE monotherapy (34 patients) and MOC31PE in combination with CsA (23 patients), was assessed. Pre- and post-treatment patient sera were analyzed in a multiplex immunoassay, and the immunogenic effects of MOC31PE were studied and in a dendritic cell maturation assay. When the data were analyzed for all treated patients regardless of cancer type, the MOC31PE alone group had a median OS of 12.7 months (95% CI = 5.6-19.8 months) compared to 6.2 months (95% CI = 5.6-6.8 months) (=.066) for the patients treated with MOC31PE + CsA group. For the subgroup of patients with colorectal cancer, the median OS survival was 16.3 months (95% CI = 5.6-27.0) for the MOC31PE only cohort ( = 15), compared to 6.0 months (CI = 5.8-6.2) ( < .001) for the combination group. The cytokine profile in patient sera and the immunological studies indicate that MOC31PE induced an immunogenic response leading to T-cell activation; a response that was suppressed in patients treated with MOC31PE + CsA. The results reveal a promising clinical benefit of anti-EpCAM immunotoxin treatment in patients with advanced disease, an effect apparently explained by a previously unknown immunogenic effect of MOC31PE.
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http://dx.doi.org/10.1080/0284186X.2019.1704864DOI Listing
April 2020

Correction to: Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy.

Cancer Immunol Immunother 2020 01;69(1):159-161

Department of Cellular Therapy, Department of Oncology, Oslo University Hospital, The Norwegian Radium Hospital, 0379, Oslo, Norway.

The original version of this article unfortunately included a mistake in Fig. 2b where the images of mice in the tumour control group (right), day 30 (bottom) should be removed as the wrong images (duplicate of day 17) were inserted by mistake. At this time point the tumour control mice were no longer alive and the images were replaced by black areas.
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http://dx.doi.org/10.1007/s00262-019-02409-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949204PMC
January 2020

Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy.

Cancer Immunol Immunother 2019 Aug 18;68(8):1235-1243. Epub 2019 Jun 18.

Department of Cellular Therapy, Department of Oncology, Oslo University Hospital, The Norwegian Radium Hospital, 0379, Oslo, Norway.

Off-target toxicity due to the expression of target antigens in normal tissue or TCR cross-reactivity represents a major risk when using T cell receptor (TCR)-engineered T cells for treatment of solid tumours. Due to the inherent cross-reactivity of TCRs it is difficult to accurately predict their target recognition pre-clinically. It has become evident that direct testing in a human being represents the best evaluation of the risks. There is, therefore, a clear unmet need for assessing the safety of a therapeutic TCR in a more controllable manner than by the injection of permanently modified cellular products. Using transiently modified T cells combined with dose escalation has already been shown feasible for chimeric antigen receptor (CAR)-engineered T cells, but nothing is yet reported for TCR. We performed a preclinical evaluation of a therapeutic TCR transiently expressed in T cells by mRNA electroporation. We analyzed if the construct was active in vitro, how long it was detectable for and if this expression format was adapted to in vivo efficacy assessment. Our data demonstrate the potential of mRNA engineered T cells, although less powerful than permanent redirection, to induce a significant response. Thus, these findings support the development of mRNA based TCR-therapy strategies as a feasible and efficacious method for evaluating TCR safety and efficacy in first-in-man testing.
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http://dx.doi.org/10.1007/s00262-019-02356-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682583PMC
August 2019

Preclinical development of CD37CAR T-cell therapy for treatment of B-cell lymphoma.

Blood Adv 2019 04;3(8):1230-1243

Division of Cancer Medicine, Department of Cellular Therapy, and.

T cells modified to express chimeric antigen receptor (CAR) targeting CD19 (CD19CAR) have produced remarkable clinical responses in patients with relapsed/refractory B-cell acute lymphoblastic leukemia. CD19CAR T-cell therapy has also demonstrated prominent effects in B-cell non-Hodgkin lymphoma (B-NHL) patients. However, a subset of patients who relapse after CD19CAR T-cell therapy have outgrowth of CD19 tumor cells. Hence, development of alternative CARs targeting other B-cell markers represents an unmet medical need for B-cell acute lymphoblastic leukemia and B-NHL. Here, we confirmed previous data by showing that, overall, B-NHL has high expression of CD37. A second-generation CD37CAR was designed, and its efficacy in T cells was compared with that of CD19CAR. In vitro assessment of cytotoxicity and T-cell function upon coculture of the CAR T cells with different target B-cell lymphoma cell lines demonstrated comparable efficacy between the 2 CARs. In an aggressive B-cell lymphoma xenograft model, CD37CAR T cells were as potent as CD19CAR T cells in controlling tumor growth. In a second xenograft model, using U2932 lymphoma cells containing a CD19 subpopulation, CD37CAR T cells efficiently controlled tumor growth and prolonged survival, whereas CD19CAR T cells had limited effect. We further show that, unlike CD19CAR, CD37CAR was not sensitive to antigen masking. Finally, CD37CAR reactivity was restricted to B-lineage cells. Collectively, our results demonstrated that CD37CAR T cells also can effectively eradicate B-cell lymphoma tumors when CD19 antigen expression is lost and support further clinical testing for patients with relapsed/refractory B-NHL.
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http://dx.doi.org/10.1182/bloodadvances.2018029678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482360PMC
April 2019

Antigen-delivery through invariant chain (CD74) boosts CD8 and CD4 T cell immunity.

Oncoimmunology 2019;8(3):1558663. Epub 2019 Jan 11.

Department of Cellular Therapy, Department of Oncology, Oslo University Hospital-Radiumhospitalet, Oslo, Norway.

Eradication of tumors by the immune system relies on the efficient activation of a T-cell response. For many years, the main focus of cancer immunotherapy has been on cytotoxic CD8 T-cell. However, stimulation of CD4 helper T cells is critical for the promotion and maintenance of immune memory, thus a good vaccine should evoke a two-dimensional T-cell response. The invariant chain (Ii) is required for the MHC class II heterodimer to be correctly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells (APC). We previously showed that by replacing the Ii CLIP peptide by an MHC-I cancer peptide, we could efficiently load MHC-I. This prompted us to test whether longer cancer peptides could be loaded on both MHC classes and whether such peptides could be accommodated in the CLIP region of Ii. We here present data showing that expanding the CLIP replacement size leads to T-cell activation. We demonstrate by using long peptides that APCs can present peptides from the same Ii molecule on both MHC-I and -II. In addition, we present evidence that antigen presentation after Ii-loading was superior to an ER-targeted minigene construct, suggesting that ER-localization was not sufficient to obtain efficient MHC-II loading. Finally, we verified that Ii-expressing dendritic cells could prime CD4 and CD8 T cells from a naïve population. Taken together our study demonstrates that CLIP peptide replaced Ii constructs fulfill some of the major requirements for an efficient vector for cancer vaccination.
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http://dx.doi.org/10.1080/2162402X.2018.1558663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350688PMC
January 2019

Combining the oncolytic peptide LTX-315 with doxorubicin demonstrates therapeutic potential in a triple-negative breast cancer model.

Breast Cancer Res 2019 01 22;21(1). Epub 2019 Jan 22.

Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, NO-0379, Oslo, Norway.

Background: Immunochemotherapy, the combined use of immunotherapy and chemotherapy, has demonstrated great promise in several cancers. LTX-315 is an oncolytic peptide with potent immunomodulatory properties designed for the local treatment of solid tumors. By inducing rapid immunogenic cell death through the release of danger-associated molecular pattern molecules (DAMPs), LTX-315 is capable of reshaping the tumor microenvironment, turning "cold" tumors "hot" through a significant increase in tumor-infiltrating lymphocytes.

Methods: We investigated the potential of LTX-315 to be used in combination with standard-of-care chemotherapy (doxorubicin, brand name CAELYX®) against triple-negative breast cancer in an orthotopic 4 T1 mammary fat pad model. Tumor growth curves were compared using one-way ANOVA analysis of variance and Tukey's multiple comparisons test, and animal survival curves were compared using the log-rank (Mantel-Cox) test. We considered p values ≤0.05 to indicate statistical significance.

Results: We found that LTX-315 displayed a strong additive antitumoral effect when used in combination with CAELYX®, and induced immune-mediated changes in the tumor microenvironment, followed by complete regression in the majority of animals treated. Furthermore, imaging techniques and histological examination showed that the combination induced strong local necrosis, followed by an increase in the infiltration of CD4+ and CD8+ immune cells into the tumor parenchymal tissue.

Conclusions: Our data demonstrate that LTX-315 is a promising combination partner with CAELYX® for the treatment of triple-negative breast cancer.
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http://dx.doi.org/10.1186/s13058-018-1092-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343247PMC
January 2019

NK cells specifically TCR-dressed to kill cancer cells.

EBioMedicine 2019 Feb 18;40:106-117. Epub 2019 Jan 18.

Department of Cellular Therapy, Department for Cancer Therapy, Oslo University Hospital Radiumhospitalet, Oslo 0379, Norway. Electronic address:

Background: Adoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome.

Methods: We herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells.

Findings: This is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells.

Interpretation: These results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution. FUND: This work was supported by the Research Council of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the University of Lausanne.
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http://dx.doi.org/10.1016/j.ebiom.2019.01.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413353PMC
February 2019

Human c-SRC kinase (CSK) overexpression makes T cells dummy.

Cancer Immunol Immunother 2018 Apr 16;67(4):525-536. Epub 2017 Dec 16.

Section for Cellular Therapy, Department for Cancer Treatment, Oslo University Hospital-Radiumhospitalet, PO Box 4953, Nydalen, 0424, Oslo, Norway.

Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide-MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells "dummy T cells" and propose to use them for safety validation of new TCRs prior to therapy.
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http://dx.doi.org/10.1007/s00262-017-2105-9DOI Listing
April 2018

The Results of Stricter Inclusion Criteria in an Immunomagnetic Detection Study of Micrometastatic Cells in Bone Marrow of Uveal Melanoma Patients - Relevance for Dormancy.

Pathol Oncol Res 2019 Jan 2;25(1):255-262. Epub 2017 Nov 2.

Department of Tumor Biology, Oslo University Hospital HF and University of Oslo, Oslo, Norway.

Approximately 50% of uveal melanoma patients develop metastases. We want to evaluate the effect of stricter criteria on our data from our previous study correlating survival and bone marrow (BM) micrometastasis results using our immunomagnetic separation (IMS) method. Mononuclear cell fractions (MNC) isolated from BM were examined for tumour cells and the patients were classified as BM positive (BM+) or BM negative (BM-). The study originally included 328 consecutive patients with uveal melanoma from 1997 to 2006. The cohort was limited to 217 patients when we introduced cyto- or histopathological verification of melanoma cells in the patient as a main new criterion for inclusion. Tumour cells were found in BM-samples in 38.7% (95% CI, 32-45) at enrolment. Until the latest work-up 43.8% (95% CI, 38-50) of patients had developed melanoma metastases. After a minimum follow-up time of 8.5 years, 60.4% (95% CI, 54-66) of patients had died. The causes were: melanoma metastases 69.5%, another type of cancer 5.4% and non-cancerous causes 19.5%. Overall median survival was shorter for the BM- patients (11.3 years) (95% CI, 10-12) compared to the BM+ (16.5 years) (95% CI, 12-14), p = 0.04, log rank test. All-cause mortality and specific melanoma mortality estimated after 12 year follow-up showed a highly significant difference comparing BM- and BM+, p = 0.010 and p = 0,017, respectively. IMS yields a high fraction of BM+ samples due to micrometastasis at diagnosis and these cells appear to have a positive prognostic impact strengthening our previous report. The late recurrences support the concept of tumour dormancy.
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http://dx.doi.org/10.1007/s12253-017-0355-7DOI Listing
January 2019

Oncolytic peptide LTX-315 induces an immune-mediated abscopal effect in a rat sarcoma model.

Oncoimmunology 2017;6(8):e1338236. Epub 2017 Jun 16.

Lytix Biopharma, Gaustadalléen 21, NO-0349 Oslo, Norway.

LTX 315 is an oncolytic peptide with potent immunological properties. In the present study, we demonstrate that intratumoral treatment with LTX-315 resulted in a complete regression and systemic immune response in a rat fibrosarcoma model. The treatment was T-cell dependent, and also resulted in an abscopal effect as demonstrated by the regression of distal non-treated lesions. Significant infiltration of CD8 T cells was observed in both treated and non-treated lesions, as shown by immunohistochemical and flow cytometric analysis. LTX-315 rapidly killed the cells with a lytic mode of action followed by the subsequent release of Danger-Associated Molecular Pattern (DAMP) molecules such as HMGB1, ATP and Cytochrome c. Together, our data demonstrate that LTX-315 represents a new approach to cancer immunotherapy, which has the potential as a novel immunotherapeutic agent.
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http://dx.doi.org/10.1080/2162402X.2017.1338236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593701PMC
June 2017

A TCR-based Chimeric Antigen Receptor.

Sci Rep 2017 09 6;7(1):10713. Epub 2017 Sep 6.

Department for Cellular Therapy, Department for Cancer Therapy, Oslo University Hospital Radiumhospitalet, Oslo, Norway.

Effector T cells equipped with engineered antigen receptors specific for cancer targets have proven to be very efficient. Two methods have emerged: the Chimeric Antigen Receptors (CARs) and T-cell Receptor (TCR) redirection. Although very potent, CAR recognition is limited to membrane antigens which represent around 1% of the total proteins expressed, whereas TCRs have the advantage of targeting any peptide resulting from cellular protein degradation. However, TCRs depend on heavy signalling machinery only present in T cells which restricts the type of eligible therapeutic cells. Hence, an introduced therapeutic TCR will compete with the endogenous TCR for the signalling proteins and carries the potential risk of mixed dimer formation giving rise to a new TCR with unpredictable specificity. We have fused a soluble TCR construct to a CAR-signalling tail and named the final product TCR-CAR. We here show that, if expressed, the TCR-CAR conserved the specificity and the functionality of the original TCR. In addition, we demonstrate that TCR-CAR redirection was not restricted to T cells. Indeed, after transduction, the NK cell line NK-92 became TCR positive and reacted against pMHC target. This opens therapeutic avenues combing the killing efficiency of NK cells with the diversified target recognition of TCRs.
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http://dx.doi.org/10.1038/s41598-017-11126-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587706PMC
September 2017

MtDNA depletion influences the transition of CD44 subtypes in human prostate cancer DU145 cells.

Tumour Biol 2017 Aug;39(8):1010428317713671

1 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital and University of Oslo, Oslo, Norway.

Our earlier study revealed that long-term ethidium bromide application causes mitochondrial DNA depletion in human prostate cancer DU145 cell line (DU145), and this DU145 subline appears to have expanded CD44 cell population than its parental wild type DU145 cells (DU145). Increasing evidence suggests that CD44 cells are highly cancer stem cell like, but it is not clear about their dynamic transition between CD44 and CD44 phenotypes in prostate cancer cells, and how it is affected by mitochondrial DNA depletion. To address these questions, four cell subpopulations were isolated from both DU145 and DU145 cell lines based on their CD44 expression level and mitochondrial membrane potential. The cell motility and colony formation capability of the fluorescence activated cell sorting-sorted cell subpopulations were further examined. It was discovered in the DU145 cells that CD44 cells could transit into both CD44 and CD44 phenotypes and that CD44 cells were prone to sustain their CD44 phenotype as renewal. However, such transition principle was altered in the DU145 cells, in which CD44 cells showed similar capability to sustain a CD44 phenotype, while the transition of CD44 cells to CD44 were suppressed. It is concluded that mitochondrial DNA depletion in the human prostate cancer DU145 cells influences their renewal and CD44 subphenotype transition. Such alterations may be the driving force for the enrichment of CD44 DU145 cells after the mitochondrial DNA depletion, although the molecular mechanisms remain unclear.
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http://dx.doi.org/10.1177/1010428317713671DOI Listing
August 2017

Human Adipose-Derived Mesenchymal Stem Cells Respond to Short-Term Hypoxia by Secreting Factors Beneficial for Human Islets In Vitro and Potentiate Antidiabetic Effect In Vivo.

Cell Med 2017 14;9(3):103-116. Epub 2017 Apr 14.

Department of Transplant Medicine, Oslo University Hospital, Oslo, Norway.

Adipose-derived mesenchymal stem cells (ASCs) release factors beneficial for islets in vitro and protect against hyperglycemia in rodent models of diabetes. Oxygen tension has been shown to induce metabolic changes and alter ASCs' release of soluble factors. The effects of hypoxia on the antidiabetic properties of ASCs have not been explored. To investigate this, we incubated human ASCs for 48 h in 21% (normoxia) or 1% O (hypoxia) and compared viability, cell growth, surface markers, differentiation capability, and soluble factors in the conditioned media (CM). Human islets were exposed to CM from ASCs incubated in either normoxia or hypoxia, and islet function and apoptosis after culture with or without proinflammatory cytokines were measured. To test hypoxic preconditioned ASCs' islet protective effects in vivo, ASCs were incubated for 48 h in normoxia or hypoxia before being injected into Balb/c Rag 1 immunodeficient mice with streptozotocin-induced insulitis. Progression of diabetes and insulin content of pancreas were measured. We found that incubation in hypoxia was well tolerated by ASCs and that levels of VEGF-A, FGF-2, and bNGF were elevated in CM from ASCs incubated in hypoxia compared to normoxia, while levels of HGF, IL-8, and CXCL1 were reduced. CM from ASCs incubated in hypoxia significantly improved human islet function and reduced apoptosis after culture, and reduced cytokine-induced apoptosis. In our mouse model, pancreas insulin content was higher in both groups receiving ASCs compared to control, but the mice receiving preconditioned ASCs had lower random and fasting blood glucose, as well as improved oral glucose tolerance compared to untreated mice. In conclusion, our in vitro results indicate that the islet protective potential of ASCs improves in hypoxia, and we give insight into factors involved in this. Finally we show that hypoxic preconditioning potentiates ASCs' antidiabetic effect in vivo.
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http://dx.doi.org/10.3727/215517917X693401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509020PMC
April 2017

Metabolic reprogramming is associated with flavopiridol resistance in prostate cancer DU145 cells.

Sci Rep 2017 07 11;7(1):5081. Epub 2017 Jul 11.

Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway.

Flavopiridol (FP) is a pan-cyclin dependent kinase inhibitor, which shows strong efficacy in inducing cancer cell apoptosis. Although FP is potent against most cancer cells in vitro, unfortunately it proved less efficacious in clinical trials in various aggressive cancers. To date, the molecular mechanisms of the FP resistance are mostly unknown. Here, we report that a small fraction human prostate cancer DU145 cells can survive long-term FP treatment and emerge as FP-resistant cells (DU145). These DU145 cells show accumulated mitochondrial lesions with stronger glycolytic features, and they proliferate in slow-cycling and behave highly migratory with strong anti-apoptotic potential. In addition, the cells are less sensitive to cisplatin and docetaxel-induced apoptotic pressure, and over-express multiple stem cell associated biomarkers. Our studies collectively uncover for the first time that FP-resistant prostate cancer cells show metabolic remodeling, and the metabolic plasticity might be required for the FP resistance-associated cancer cell stemness up-regulation.
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http://dx.doi.org/10.1038/s41598-017-05086-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506068PMC
July 2017

T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces tumor growth.

Oncoimmunology 2017;6(4):e1302631. Epub 2017 Mar 17.

Section for Cancer Immunology, Institute for Cancer Research, Oslo University Hospital-The Norwegian Radium Hospital, Oslo, Norway.

T-cell receptor (TCR) transfer is an attractive strategy to increase the number of cancer-specific T cells in adoptive cell therapy. However, recent clinical and pre-clinical findings indicate that careful consideration of the target antigen is required to limit the risk of off-target toxicity. Directing T cells against mutated proteins such as frequently occurring frameshift mutations may thus be a safer alternative to tumor-associated self-antigens. Furthermore, such frameshift mutations result in novel polypeptides allowing selection of TCRs from the non-tolerant T-cell repertoire circumventing the problem of low affinity TCRs due to central tolerance. The transforming growth factor β Receptor II frameshift mutation (TGFβRII) is found in Lynch syndrome cancer patients and in approximately 15% of sporadic colorectal and gastric cancers displaying microsatellite instability (MSI). The -1A mutation within a stretch of 10 adenine bases (nucleotides 709-718) of the TGFβRII gene gives rise to immunogenic peptides previously used for vaccination of MSI+ colorectal cancer patients in a Phase I clinical trial. From a clinically responding patient, we isolated a cytotoxic T lymphocyte (CTL) clone showing a restriction for HLA-A2 in complex with TGFβRII peptide. Its TCR was identified and shown to redirect T cells against colon carcinoma cell lines harboring the frameshift mutation. Finally, T cells transduced with the HLA-A2-restricted TGFβRII-specific TCR were demonstrated to significantly reduce the growth of colorectal cancer and enhance survival in a NOD/SCID xenograft mouse model.
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http://dx.doi.org/10.1080/2162402X.2017.1302631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414866PMC
March 2017

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

Exp Cell Res 2017 03 9;352(2):218-224. Epub 2017 Feb 9.

Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo.
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http://dx.doi.org/10.1016/j.yexcr.2017.02.005DOI Listing
March 2017

T-helper cell receptors from long-term survivors after telomerase cancer vaccination for use in adoptive cell therapy.

Oncoimmunology 2016;5(12):e1249090. Epub 2016 Oct 24.

Department of Haematology, UCL Cancer Institute, University College London , London, UK.

We herein report retargeting of T-helper (Th) cells against the universal cancer antigen telomerase for use in adoptive cell therapy. The redirected Th cells may counter tumor tolerance, transform the inflammatory milieu, and induce epitope spreading and cancer senescence. We have previously conducted a series of trials evaluating vaccination with telomerase peptides. From long-term survivors, we isolated >100 CD4 Th-cell clones recognizing telomerase epitopes. The clones were characterized with regard to HLA restriction, functional avidity, fine specificity, proliferative capacity, cytokine profile, and recognition of naturally processed epitopes. DP4 is the most prevalent HLA molecule worldwide. Two DP4-restricted T-cell clones with different functional avidity, C13 and D71, were selected for molecular T-cell receptor (TCR) cloning. Both clones showed a high proliferative capacity, recognition of naturally processed telomerase epitopes, and a polyfunctional and Th1-weighted cytokine profile. TCR C13 and D71 were cloned into the retroviral vector MP71 together with the compact and GMP-applicable marker/suicide gene RQR8. Both TCRs were expressed well in recipient T cells after PBMC transduction. The transduced T cells co-expressed RQR8 and acquired the desired telomerase specificity, with a polyfunctional response including production of TNFa, IFNγ, and CD107a. Interestingly, the DP4-restricted TCRs were expressed and functional both in CD4 and CD8 T cells. The findings demonstrate that the cloned TCRs confer recipient T cells with the desired hTERT-specificity and functionality. We hypothesize that adoptive therapy with Th cells may offer a powerful novel approach for overcoming tumor tolerance and synergize with other forms of immunotherapy.
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http://dx.doi.org/10.1080/2162402X.2016.1249090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214348PMC
October 2016

Immune response and long-term clinical outcome in advanced melanoma patients vaccinated with tumor-mRNA-transfected dendritic cells.

Oncoimmunology 2016;5(11):e1232237. Epub 2016 Oct 18.

Department for Cell Therapy, Radiumhospitalet, Oslo University Hospital , Oslo, Norway.

The most effective anticancer immune responses are probably directed against patient-specific neoantigens. We have developed a melanoma vaccine targeting this individual mutanome based on dendritic cells (DCs) loaded with autologous tumor-mRNA. Here, we report a phase I/II trial evaluating toxicity, immune response and clinical outcome in 31 metastatic melanoma patients. The first cohort (n = 22) received the vaccine without any adjuvant; the next cohort (n = 9) received adjuvant IL2. Each subject received four weekly intranodal or intradermal injections, followed by optional monthly vaccines. Immune response was evaluated by delayed-type hypersensitivity (DTH), T cell proliferation and cytokine assays. Data were collected for 10 y after inclusion of the last patient. No serious adverse events were detected. In the intention-to-treat-cohort, we demonstrated significantly superior survival compared to matched controls from a benchmark meta-analysis (1 y survival 43% vs. 24%, 2 y 23% vs. 6.6%). A tumor-specific immune response was demonstrated in 16/31 patients. The response rate was higher after intradermal than intranodal vaccination (80% vs. 38%). Immune responders had improved survival compared to non-responders (median 14 mo vs. 6 mo; = 0.030), and all eight patients surviving >20 mo were immune responders. In addition to the tumor-specific response, most patients developed a response against autologous DC antigens. The cytokine profile was polyfunctional and did not follow a Th1/Th2 dichotomy. We conclude that the favorable safety profile and evidence of a possible survival benefit warrant further studies of the RNA/DC vaccine. The vaccine appears insufficient as monotherapy, but there is a strong rationale for combination with checkpoint modulators.
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http://dx.doi.org/10.1080/2162402X.2016.1232237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5139630PMC
October 2016

Cost and clinical outcome of islet transplantation in Norway 2010-2015.

Clin Transplant 2017 01 9;31(1). Epub 2016 Dec 9.

Department of Transplant Medicine, Cancer Institute, Oslo University Hospital, Oslo, Norway.

Islet transplantation is a minimally invasive β-cell replacement strategy. Islet transplantation is a reimbursed treatment in Norway. Here, we summarize the cost and clinical outcome of 31 islet transplantations performed at Oslo University Hospital (OUS) from January 2010 to June 2015. Patients were retrospectively divided into three groups. Thirteen patients received either one or two islet transplantation alone (ITA), while five patients received islet transplantation after previous solid organ transplantation. For the group receiving 2 ITA, Kaplan-Meier estimates show an insulin independence of 20% more than 4 years after their last transplantation. An estimated 70% maintain at least partial graft function, defined as fasting C-peptide >0.1 nmol L , and 47% maintain a HbA1c below 6.5% or 2 percent points lower than before ITA. For all groups combined, we estimate that 44% of the patients have a 50% reduction in insulin requirement 4 years after the initial islet transplantation. The average cost for an islet transplantation procedure was 347 297±60 588 NOK, or 35 424±6182 EUR, of which isolation expenses represent 34%. We hereby add to the common pool of growing experience with islet transplantation and also describe the cost of the treatment at our center.
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http://dx.doi.org/10.1111/ctr.12871DOI Listing
January 2017

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features.

Oncotarget 2016 Jun;7(26):40297-40313

Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, 0379, Norway.

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features.
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http://dx.doi.org/10.18632/oncotarget.9610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130009PMC
June 2016

Diversification of Antitumour Immunity in a Patient with Metastatic Melanoma Treated with Ipilimumab and an IDO-Silenced Dendritic Cell Vaccine.

Case Rep Med 2016 18;2016:9639585. Epub 2016 Jul 18.

Department of Cell Therapy, Oslo University Hospital, Montebello, 0310 Oslo, Norway.

Indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells (DCs) inhibits T-cell activation and promotes T-cell differentiation into regulatory T-cells. Moreover, IDO expression promotes resistance to immunotherapies targeting immune checkpoints such as the cytotoxic T lymphocyte antigen-4 (CTLA-4). Here, a patient with metastatic melanoma pretreated with ipilimumab, an anti-CTLA-4 blocking antibody, was vaccinated with IDO-silenced DCs cotransfected with mRNA for survivin or hTERT tumour antigens. During vaccination, T-cell responses to survivin and hTERT tumour antigens were generated, and a certain degree of clinical benefit was achieved, with a significant reduction in lung, liver, and skin metastases, along with a better performance status. T-cell responses against MART-1 and NY-ESO-1 tumour antigens were also detected in the peripheral blood. The patient also mounted an antibody response to several melanoma proteins, indicating diversification of the antitumour immunity in this patient. The identification of such serum antibody-reacting proteins could facilitate the discovery of tumour neoantigens.
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http://dx.doi.org/10.1155/2016/9639585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967686PMC
August 2016

ILs-3, 6 and 11 increase, but ILs-10 and 24 decrease stemness of human prostate cancer cells in vitro.

Oncotarget 2015 Dec;6(40):42687-703

Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Henan, China.

Cancer stem cells (CSCs) are associated with cancer recurrence and metastasis. Prostate cancer cells often metastasize to the bone with a complex microenvironment of cytokines favoring cell survival. In this study, the cell stemness influence of a group of interleukins including IL-3, 6, 10, 11 and 24 on human prostate cancer cell lines LNCaP and PC-3 was explored in vitro. Sulforhodamine B(SRB) and 5-ethynyl-2'-deoxyuridine (EdU) assays were applied to examine the effect on cell proliferation, and wound healing and transwell assays were used for migration and invasion studies, in addition to colony formation, Western blotting and flowcytometry for the expression of stemness factors and chemotherapy sensitivity. We observed that ILs-3, 6 and 11 stimulated while ILs-10 and 24 inhibited the growth, invasion and migration of both cell lines. Interestingly, ILs-3, 6 and 11 significantly promoted colony formation and increased the expression of SOX2, CD44 and ABCG2 in both prostate cancer cell lines. However, ILs-10 and 24 showed the opposite effect on the expression of these factors. In line with the above findings, treatment with either IL-3 or IL-6 or IL-11 decreased the chemosensitivity to docetaxel while treatment with either IL-10 or IL-24 increased the sensitivity of docetaxel chemotherapy. In conclusion, our results suggest that ILs-3, 6 and 11 function as tumor promoters while ILs-10 and 24 function as tumor suppressors in the prostate cancer cell lines PC-3 and LNCaP in vitro, and such differences may attribute to their different effect on the stemness of PCa cells.
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http://dx.doi.org/10.18632/oncotarget.5883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767463PMC
December 2015

Immunomagnetic detection of micrometastatic cells in bone marrow of uveal melanoma patients: a paradox.

Acta Ophthalmol 2015 Feb;93(1):59-66

Eye Department, University Hospital HF, Oslo, Norway.

Purpose: Our objective was to study survival rates with the bone marrow (BM) results in a cohort of uveal melanoma patients with long follow-up.

Methods: Mononuclear cell fractions isolated from BM were examined for tumour cells using our immunomagnetic separation (IMS) method. The patients were classified as BM positive or BM negative. Clinical follow-up, histopathological findings, vital status and cause of death were registered.

Results: The study included 328 consecutive patients with uveal melanoma from 1997 to 2006. Tumour cells were found in BM samples in 29% (95% CI, 25-34) at enrolment (96 cases). After a minimum follow-up time of 6 years, 156 (48%) (95% CI, 42-53) melanoma patients had died. The causes were as follows: melanoma metastases 92 (59%), another cancer 20 (13%) and non-cancer 44 (28%). Nine patients were still living with melanoma metastases. Until the latest work-up, 101(31%) (95% CI, 26-36) patients had developed melanoma metastases. Cyto- or histopathological verification of the metastatic lesions was obtained in 85 cases (84%). In the group with melanoma metastases, 28 tested BM positive at study entry (28%) (95% CI, 19-38). In total, 39 of 101 with metastases tested positive at least once after a maximum of three tests (39%) (95% CI, 29-49). The overall median survival from the first BM test was shorter for the BM negative patients (9.5 years) compared with the BM positive (14.4 years), p = 0.02, log rank test.

Conclusion: Ocular melanoma cells detected in BM seem to have a positive prognostic impact on survival in contrast to our original hypothesis.
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http://dx.doi.org/10.1111/aos.12462DOI Listing
February 2015

Sequential intranodal immunotherapy induces antitumor immunity and correlated regression of disseminated follicular lymphoma.

Blood 2015 Jan 7;125(1):82-9. Epub 2014 Oct 7.

K. G. Jebsen Center for Cancer Immunotherapy, Institute for Clinical Medicine, University of Oslo, Oslo, Norway; Department of Immunology.

Advanced stage follicular lymphoma (FL) is incurable by conventional therapies. In the present pilot clinical trial, we explored the efficacy and immunogenicity of a novel in situ immunotherapeutic strategy. Fourteen patients with untreated or relapsed stage III/IV FL were included and received local radiotherapy to solitary lymphoma nodes and intranodal injections of low-dose rituximab (5 mg), immature autologous dendritic cells, and granulocyte-macrophage colony-stimulating factor at the same site. The treatment was repeated 3 times targeting different lymphoma nodes. Primary end points were clinical responses and induction of systemic immunity. Five out of 14 patients (36%) displayed objective clinical responses, including 1 patient with cutaneous FL who showed regression of skin lesions. Two of the patients had durable complete remissions. Notably, the magnitude of vaccination-induced systemic CD8 T-cell-mediated responses correlated closely with reduction in total tumor area (r = 0.71, P = .006), and immune responders showed prolonged time to next treatment. Clinical responders did not have a lower tumor burden than nonresponders pretreatment, suggesting that the T cells could eliminate large tumor masses once immune responses were induced. In conclusion, the combined use of 3 treatment modalities, and in situ administration in single lymphoma nodes, mediated systemic T-cell immunity accompanied by regression of disseminated FL. The trial was registered at www.clinicaltrials.gov as #NCT01926639.
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http://dx.doi.org/10.1182/blood-2014-07-592162DOI Listing
January 2015
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