| 2. |
- Hojjat-Farsangi, Mohammad, et al.
(författare)
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The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL) cells
- 2013
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Ingår i: PLOS ONE. - Stockholm : Karolinska Institutet, Dept of Oncology-Pathology. - 1932-6203.
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Tidskriftsartikel (refereegranskat)abstract
- Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.
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| 3. |
- Palma, Marzia
(författare)
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Immunological and molecular studies for the development of vaccine therapy for chronic lymphocytic leukemia
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chronic lymphocytic leukaemia (CLL) is a malignant lymphoproliferative disorder which typically affects elderly people. It is the commonest leukemia in the Western adults, accounting for 25-30% of all leukemias and for 10% of all hematological neoplasms. Although new modalities such as combination therapy with fludarabine, cyclophosphamide (CTX) and the anti-CD20 antibody rituximab have greatly improved clinical outcome in a fraction of patients, CLL is largely considered incurable and there is a continuous need to develop new treatment strategies. Anti-cancer active specific immunotherapy aims at activating the patient's immune system to recognize and eliminate the tumor. A number of clinical observations as well as several preclinical studies indicate that CLL is responsive to immune effector functions. In the first part of this thesis, we investigated the ability of a promiscuous HLA class II epitope, hTERT (611 626) (GV1001) to elicit antileukemic immune responses in vitro. We demonstrated that CLL patients with hTERT-expressing leukemic cells have naturally occurring hTERT-specific T cells that proliferate and can be expanded in vitro and used to lyse autologous CLL cells. We therefore identified telomerase as a vaccine candidate in CLL. We then analyzed hTERT mRNA splicing patterns in CLL by a newly designed quantitative PCR assay and showed that the expression of the functional transcript of hTERT (hTERT-FL) is independent from disease phase in IgHV mutated but not in unmutated patients. This finding highlights the necessity of focusing on this transcript when analyzing hTERT expression and encourages further studies to assess whether hTERT-FL could generate novel epitopes that may serve as immunotherapy targets. In the second part of the thesis, we studied safety, immune and clinical effects of vaccination with autologous DC loaded with apoptotic CLL cells (Apo-DC) in CLL patients in a phase I clinical trial. Using a combination of leukapheresis and affinity-based technologies (CliniMACS®) for monocyte enrichment, we were able to produce a sufficient amount of DC vaccine that met accepted and established quality criteria. Sixteen patients were accrued stepwise in three different cohorts receiving Apo-DC alone, Apo-DC + granulocyte-macrophage-colony-stimulating-factor (GM-CSF), or Apo-DC + GM-CSF + low-dose CTX. Vaccination was well tolerated and increased leukemia-specific immunity in 10/15 (66%) of the patients (2/5, 3/5 and 5/5 in the three cohorts, respectively). No significant difference in time-to progression (TTP) between immuneresponders and non-immune responders was observed. An additional patient was immunized repeatedly for a long period of time and achieved a complete response in blood and a nodular partial response in bone marrow. CD4+CD25highFOXP3+ regulatory T-cells (Tregs) measured in one year follow-up period were significantly lower in immuneresponders vs non-responders (p<0.0001). In this study, we demonstrated that vaccination with Apo-DC is a feasible approach that can generate immune responses and potentially clinical responses and that combination with GM-CSF and low-dose CTX functions as an immunological adjuvant in this setting. In conclusion, the studies presented in this thesis suggest that immunotherapy is a promising approach in CLL and promote further investigation to better define the vaccination strategy and combination with immune enhancing/modulating drugs which holds the greatest potential to generate immune responses and clinical benefit in CLL patients.
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