Development of tumor-reactive T cells after nonmyeloablative allogeneic hematopoietic stem cell transplant for chronic lymphocytic leukemia.

PURPOSE: Allogeneic nonmyeloablative hematopoietic stem cell transplant (NM-HSCT) can result in durable remission of chronic lymphocytic leukemia (CLL). It is thought that the efficacy of NM-HSCT is mediated by recognition of tumor cells by T cells in the donor stem cell graft. We evaluated the development of CTLs specific for CLL after NM-HSCT to determine if their presence correlated with antitumor efficacy. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells obtained from 12 transplant recipients at intervals after NM-HSCT were stimulated in vitro with CLL cells. Polyclonal T-cell lines and CD8(+) T-cell clones were derived from these cultures and evaluated for lysis of donor and recipient target cells including CLL. The presence and specificity of responses was correlated with clinical outcomes. RESULTS: Eight of the 12 patients achieved remission or a major antitumor response and all 8 developed CD8(+) and CD4(+) T cells specific for antigens expressed by CLL. A clonal analysis of the CD8(+) T-cell response identified T cells specific for multiple minor histocompatibility (H) antigens expressed on CLL in six of the responding patients. A significant fraction of the CD8(+) T-cell response in some patients was also directed against nonshared tumor-specific antigens. By contrast, CLL-reactive T cells were not detected in the four patients who had persistent CLL after NM-HSCT, despite the development of graft-versus-host disease. CONCLUSIONS: The development of a diverse T-cell response specific for minor H and tumor-associated antigens expressed by CLL predicts an effective graft-versus-leukemia response after NM-HSCT

Comprehensive evaluation of methods to assess overall and cell-specific immune infiltrates in breast cancer

Background: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. Methods: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. Results: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infilt

Therapeutic Potential and Challenges of Targeting Receptor Tyrosine Kinase ROR1 with Monoclonal Antibodies in B-Cell Malignancies

Based on its selective cell surface expression in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), receptor tyrosine kinase ROR1 has recently emerged as a promising target for therapeutic monoclonal antibodies (mAbs). To further assess the suitability of ROR1 for targeted therapy of CLL and MCL, a panel of mAbs was generated and its therapeutic utility was investigated.A chimeric rabbit/human Fab library was generated from immunized rabbits and selected by phage display. Chimeric rabbit/human Fab and IgG1 were investigated for their capability to bind to human and mouse ROR1, to mediate antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and internalization, and to agonize or antagonize apoptosis using primary CLL cells from untreated patients as well as MCL cell lines. A panel of mAbs demonstrated high affinity and specificity for a diverse set of epitopes that involve all three extracellular domains of ROR1, are accessible on the cell surface, and mediate internalization. The mAb with the highest affinity and slowest rate of internalization was found to be the only mAb that mediated significant, albeit weak, ADCC. None of the mAbs mediated CDC. Alone, they did not enhance or inhibit apoptosis.Owing to its relatively low cell surface density, ROR1 may be a preferred target for armed rather than naked mAbs. Provided is a panel of fully sequenced and thoroughly characterized anti-ROR1 mAbs suitable for conversion to antibody-drug conjugates, immunotoxins, chimeric antigen receptors, and other armed mAb entities for preclinical and clinical studies

Active-site structure, binding and redox activity of the heme–thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study

Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6

Supplementary Appendix. All-trans retinoic acid works synergistically with the γ- secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T cells

Supplement Figure 1: ATRA treatment does not affect the viability of myeloma cell lines. MM.1S, OPM-2 and NCI-H929 cells were treated with ATRA for up to 72 hours. Cell viability was measured by flow cytometry and 7AAD staining (n=6). Bar diagrams show mean values +SD.Supplement Figure 2: ATRA plus crenigacestat treatment enhance BCMA expression on myeloma cell lines. Bar diagram shows BCMA expression on OPM-2 cells (n=3) after treatment with 100 nM ATRA and/or 10 nM GSI crenigacestat for 72 hours. Bar diagram shows mean values +SD. P-values between indicated groups were calculated using unpaired t-test. *p<0.05, **p<0.01.Supplement Figure 3: ATRA treatment leads to increased BCMA transcripts in OPM-2 myeloma cells. BCMA RNA levels in OPM-2 were analyzed by quantitative reverse transcription PCR (qRT-PCR) assay after incubation with increasing doses of ATRA for 48 hours (n=3). Bar diagram shows mean values +SD. P-values between indicated groups were calculated using unpaired t-test. *p<0.05.Supplement Figure 4: ATRA treatment leads to enhanced BCMA expression on primary myeloma cells. Representative flow cytometric analysis of BCMA expression on primary myeloma cells that had been cultured in the absence or presence of ATRA at different concentrations for 72 hours. 7-AAD was used to exclude dead cells from analysis.Supplement Figure 5: ATRA treatment does not impair viability of primary myeloma cells. Viability of primary myeloma cells with or without 72 hours of ATRA treatment was analyzed by flow cytometry and 7-AAD staining (n=5 biological replicates). Bar diagram shows mean values +SD.Supplement Figure 6: sBCMA does not impair BCMA CAR T cell functionality. CD8+ BCMA-CAR T-cells were co-cultured with MM.1S target cells in absence or presence of 150 ng/ml of soluble BCMA. After 4 hours, cytotoxicity was evaluated by bioluminescence- based assay. Diagram shows mean values +/-SD.Supplement Figure 7: ATRA treatment does not increase shedding of sBCMA. sBCMA concentration in the supernatant of OPM-2 and NCI-H929 after incubation with increasing doses of ATRA was analyzed by ELISA. Cell lines were cultured at 1x106/well (n=3 technical replicates). Bar diagrams show mean values +SD, P-values between indicated groups were calculated using 2way ANOVA. n.s. = not significant, *p<0.05, **p<0.01.Supplement Figure 8: BCMA-CAR T-cells confer enhanced cytotoxicity against ATRA plus crenigacestat-treated OPM-2 cells in vitro. OPM-2 cells were incubated with 100 nM ATRA and/or 10 nM GSI for 72 hours or were left untreated. Cytolytic activity of CD8+ BCMA- CAR T-cells was determined in a bioluminescence-based assay after 4h of co-incubation with target cells. Assay was performed in triplicate wells with 5,000 target cells per well. Data are presented as mean values +SD (n=4 biological replicates). P-values between indicated groups were calculated using unpaired t-test. n.s. = not significant, *p<0.05.Supplement Figure 9: Patient-derived BCMA-CAR T-cells confer enhanced cytotoxicity against ATRA-treated MM.1S cells. MM.1S cells were incubated with 50 nM ATRA for 72 hours or were left untreated. Cytolytic activity of MM patient-derived CD8+ BCMA-CAR T-cells was determined in a bioluminescence-based assay after 4h of co-incubation with target cells. Data are presented as mean values +SD of triplicate wells. P-values between indicated groups were calculated using unpaired t-test. *p<0.05, **p<0.01.Peer reviewe

Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant

Medical Therapies for Uterine Fibroids - A Systematic Review and Network Meta-Analysis of Randomised Controlled Trials

BACKGROUND: Uterine fibroids are common, often symptomatic and a third of women need repeated time off work. Consequently 25% to 50% of women with fibroids receive surgical treatment, namely myomectomy or hysterectomy. Hysterectomy is the definitive treatment as fibroids are hormone dependent and frequently recurrent. Medical treatment aims to control symptoms in order to replace or delay surgery. This may improve the outcome of surgery and prevent recurrence. PURPOSE: To determine whether any medical treatment can be recommended in the treatment of women with fibroids about to undergo surgery and in those for whom surgery is not planned based on currently available evidence. STUDY SELECTION: Two authors independently identified randomised controlled trials (RCT) of all pharmacological treatments aimed at the treatment of fibroids from a list of references obtained by formal search of MEDLINE, EMBASE, Cochrane library, Science Citation Index, and ClinicalTrials.gov until December 2013. DATA EXTRACTION: Two authors independently extracted data from identified studies. DATA SYNTHESIS: A Bayesian network meta-analysis was performed following the National Institute for Health and Care Excellence-Decision Support Unit guidelines. Odds ratios, rate ratios, or mean differences with 95% credible intervals (CrI) were calculated. RESULTS AND LIMITATIONS: A total of 75 RCT met the inclusion criteria, 47 of which were included in the network meta-analysis. The overall quality of evidence was very low. The network meta-analysis showed differing results for different outcomes. CONCLUSIONS: There is currently insufficient evidence to recommend any medical treatment in the management of fibroids. Certain treatments have future promise however further, well designed RCTs are needed

Management of adults and children receiving CAR T-cell therapy: 2021 best practice recommendations of the European Society for Blood and Marrow Transplantation (EBMT) and the Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association (EHA)

Background Several commercial and academic autologous chimeric antigen receptor T-cell (CAR-T) products targeting CD19 have been approved in Europe for relapsed/refractory B-cell acute lymphoblastic leukemia, high-grade B-cell lymphoma and mantle cell lymphoma. Products for other diseases such as multiple myeloma and follicular lymphoma are likely to be approved by the European Medicines Agency in the near future. Design The European Society for Blood and Marrow Transplantation (EBMT)-Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association collaborated to draft best practice recommendations based on the current literature to support health care professionals in delivering consistent, high-quality care in this rapidly moving field. Results Thirty-six CAR-T experts (medical, nursing, pharmacy/laboratory) assembled to draft recommendations to cover all aspects of CAR-T patient care and supply chain management, from patient selection to long-term follow-up, post-authorisation safety surveillance and regulatory issues. Conclusions We provide practical, clinically relevant recommendations on the use of these high-cost, logistically complex therapies for haematologists/oncologists, nurses and other stakeholders including pharmacists and health sector administrators involved in the delivery of CAR-T in the clinic