ALBERT

Description: Mycosis fungoides (MF) is a cutaneous T-cell lymphoma characterized by multifocal disease and protracted clinical course. The few studies that have assessed T-cell receptor (TCR) gene rearrangements (GRs) present at different anatomic sites in MF have generally reported a common clone. We used a previously validated 4-color polymerase chain reaction (PCR) assay to assess the size and V-family usage of TCR-γ GRs in 102 concurrent and/or sequential morphologically involved biopsy specimens (91 skin and 11 lymph nodes) from 39 MF patients. This assay detected TCR-γ clonal GRs in 89 samples (87%) from 36 patients (92%). In 24 patients (77%), an identical clonal GR was present in at least 2 skin samples. However, in one third of these patients, additional different clonal GRs were also noted. Four patients (13%) had clonal GRs that were distinct in different skin samples. In 3 patients (10%), no GR was detected in any sample. In a comparison of lymph node and skin samples, 8 patients had the identical clonal GRs at both sites, 2 patients had different clonal GRs, and 1 patient had no GR identified at either site. Independent of clinical stage, patients who had the same GR detected in multiple concurrent biopsy specimens at the time of diagnosis were more likely to have progressive disease than those who had different GRs (P = .04). Four-color TCR-γ PCR analysis can uncover multiple distinct clonal GRs in different samples consistent with multiclonal or oligoclonal disease in a significant proportion of MF patients. Demonstration of identical clonal GRs in multiple biopsy specimens at the time of diagnosis may provide prognostic information related to disease progression.

Description: Fetal/neonatal immune responses generally are considered to be immature and weaker than that of adults. We have studied the cord-blood T cells of newborns congenitally infected with Trypanosoma cruzi, the protozoan agent of Chagas disease. Our data demonstrate a predominant activation of CD8 T cells expressing activation markers and armed to mediate effector functions. The analysis of the T-cell receptor beta chain variable repertoire shows the oligoclonal expansion of these T lymphocytes, indicating that activation was driven by parasite antigens. Indeed, we have detected parasite-specific CD8 T cells secreting interferon-γ after coincubation with live T cruzi. This response is enhanced in the presence of recombinant interleukin-15, which limits the T-cell spontaneous apoptosis. These findings point out that the fetal immune system is more competent than previously appreciated, since fetuses exposed to live pathogens are able to develop an adultlike immune CD8 T-cell response.

Description: Fetal/neonatal immune responses generally are considered to be immature and weaker than that of adults. We have studied the cord-blood T cells of newborns congenitally infected with Trypanosoma cruzi, the protozoan agent of Chagas disease. Our data demonstrate a predominant activation of CD8 T cells expressing activation markers and armed to mediate effector functions. The analysis of the T-cell receptor beta chain variable repertoire shows the oligoclonal expansion of these T lymphocytes, indicating that activation was driven by parasite antigens. Indeed, we have detected parasite-specific CD8 T cells secreting interferon-γ after coincubation with live T cruzi. This response is enhanced in the presence of recombinant interleukin-15, which limits the T-cell spontaneous apoptosis. These findings point out that the fetal immune system is more competent than previously appreciated, since fetuses exposed to live pathogens are able to develop an adultlike immune CD8 T-cell response.

Description: Neutrophils are mobilized to the vascular wall during vessel inflammation. Published data are conflicting on phagocytic nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activation during the hypertensive state, and the capacity of angiotensin II (Ang II) to modulate the intracellular redox status has not been analyzed in neutrophils. We here describe that Ang II highly stimulates endogenous and extracellular O2- production in these cells, consistent with the translocation to the cell membrane of the cytosolic components of NADPH oxidase, p47phox, and p67phox. The Ang II–dependent O2- production was suppressed by specific inhibitors of AT1 receptors, of the p38MAPK and ERK1/2 pathways, and of flavin oxidases. Furthermore, Ang II induced a robust phosphorylation of p38MAPK, ERK1/2, and JNK1/2 (particularly JNK2), which was hindered by inhibitors of NADPH oxidase, tyrosine kinases, and ROS scavengers. Ang II increased cytosolic Ca2+ levels—released mainly from calcium stores—enhanced the synthesis de novo and activity of calcineurin, and stimulated the DNA-binding activity of the transcription factor NF-κB in cultured human neutrophils. Present data demonstrate for the first time a stimulatory role of Ang II in the activation of phagocytic cells, underscore the relevant role of ROS as mediators in this process, and uncover a variety of signaling pathways by which Ang II operates in human neutrophils.

Description: Several reasons warrant the development of innovative therapeutic strategies for HIV/AIDS. These include the inability of highly active antiretroviral therapy (HAART) to eradicate the virus, the HAART-induced severe long-term toxicity occurring in patients, the development of HAART-resistant HIV-1 strains in the host, and the lack of an efficacious vaccine. Genetic engineering of hematopoietic stem cells (HSC) combined with nonmyeloablative conditioning proved safety and efficacy in the treatment of adenosine deaminase-deficient severe combined immunodeficiency. The feasibility of such an approach in HIV-1 infection remains, however, to be determined. In an open-label prospective trial, 18 patients with HIV-1 infection (mean±SE age 35.7±1.2, range 18.9–40; HAART since at least 3 months; CD4+ T cell counts 〉200/μl) have been enrolled in a HSC retroviral vector gene therapy trial using RevM10 and polAS as anti-HIV genes. Nine patients received fresh transduced CD34+ cells and all study treatments, including CD34+ cell mobilisation with G-CSF (10 μg/kg/day for 5 days), CD34+ cell collection through aphaeresis, and nonmyeloablative conditioning (1.8 g/m2 cyclophosphamide [CY]), while 9 did not undergo all study phases. All patients have been followed-up for at least 48 weeks. Mean±SE baseline CD4+ T cell counts were 577±42, while plasma HIV-1 RNA levels (VL) were below the limit of detection (80 copies/ml) of the assay (Nasba Organon) in 9 out of 18 patients. CD34+ cells were efficiently mobilized and collected from patients with HIV-1 infection, achieving 4.42±0.64 x 106 CD34+ cells/kg after purification (CliniMACS, Miltenyi Biotec), and 3.93±1.2 x 106 viable CD34+ cells/kg in the infusion product, 30% of which were transduced CD34+ cells. It is worth noting that 1) effective VL suppression significantly increased the yields of mobilization, purification and transduction processes, and 2) peripheral blood CD34+ cell counts before aphaeresis (mean, 72 cells/μl) predicted the number of viable CD34+ cells infused (β 0.722, 95% CI 0.007–0.092, P=0.028, regression analysis), and a cut-off value 〉30 CD34+ cells/μl predicted the success of all procedures (P=0.018, χ2 analysis, Fisher’s exact test). Gene marking levels, predicted by the number of transduced cells infused, were detectable in all patients, though they significantly decreased over time. CY conditioning caused a marked decrease in CD4+ T cell counts, restored over long-term follow-up. This recovery correlated with levels of CD4+ TCR-rearrangement excision circles and CD4+CD45RA+CCR7+ naïve T cells, indicating thymus regeneration capacity in 〉30-year-old patients with HIV-1 infection. Importantly, CMV-specific IL-2- and IFN- γ-secreting CD4+CD69+ T cells were able to expand while no clinically relevant CMV reactivation occurred; moreover, proportions of IL-2, IL-2/IFN- γ, and IFN-γ-secreting HSV, TT, and EBV-specific CD4+ T cells were not altered by CY over time. These data indicate that effective stem cell gene transfer is feasible in patients with HIV-1 infection, and suggest the use of non-lymphocyte-toxic conditioning regimen, such as busulfan.

Description: We have reported that rituximab triggers and inhibits anti-apoptotic gene products in NHL B-cell lines resulting in sensitization to drug-induced apoptosis (Alas et al., Clin. Cancer Res.8:836, 2001; Jazirehi et al., Mol. Cancer Therapy2:1183, 2003; Vega et al., Oncogene23:3530, 2004 ). This study investigated whether rituximab also modifies intracellular signaling pathways resulting in the sensitization of NHL cells to Fas-induced apoptosis. Treatment of the NHL cell lines (2F7, Ramos, and Raji) with rituximab (20 μg/ml) sensitized the cells to CH-11 (FasL agonist mAb) -induced apoptosis and synergy was achieved. Fas expression was up-regulated by rituximab as early as 6 h post treatment as determined by flow cytometry, RT-PCR, and Western. Rituximab inhibited both the expression and activity of the transcription repressor Yin-Yang 1 (YY1) that negatively regulates Fas transcription. Inhibition of YY1 resulted in upregulation of Fas expression and sensitization of the tumor cells to CH-11-induced apoptosis. Downregulation of YY1 expression was the result of rituximab-induced inhibition of both the p38MAPK signaling pathway and constitutive NF- κB activity. The dual roles of NF-κB and YY1 in the regulation of Fas expression were corroborated by the use of a dominant-active inhibitor of NF- κB (Ramos IκB-ER mutant) and YY1 siRNA, respectively. The role of rituximab-mediated inhibition of the p38MAPK/NF- κB/YY1 pathways, which result in both Fas upregulation and sensitization to CH11-induced apoptosis, was corroborated by the use of specific chemical inhibitors directed at various targets of these pathways. Rituximab-mediated sensitization to CH-11-induced apoptosis was executed through the Type II mitochondrial apoptotic pathway. Altogether, these findings provide a novel mechanism of rituximab-mediated signaling by inhibiting the p38MAPK/NF- κB/YY1 pathways and resulting in the sensitization of B NHL to Fas-induced apoptosis. These findings may have significant clinical implications and suggest an additional mechanism of rituximab-mediated effect in vivo in addition to CDC and ADCC.

Description: Rituximab (chimeric anti-CD20 mAb) is currently being used in the therapy of Non-Hodgkin’s B-cell Lymphoma (NHL). It has also been reported that the combination of rituximab and chemotherapy is effective in patients with NHL. Several proposed mechanisms have been suggested for rituximab-mediated effects in vivo and include ADCC, CDC and apoptosis. In addition, we have reported that rituximab can trigger the tumor cells and modifies several intracellular survival signaling pathways resulting in the sensitization of tumor cells to various chemotherapeutic drugs (Alas et al., Clin. Cancer Res.8:836, 2001; Jazirehi et al., Mol. Cancer Therapy2:1183, 2003; Vega et al., Oncogene23:3530, 2004). Rituximab-mediated signaling might have resulted from directly triggering CD20 alone or in conjunction with triggering FcRs on the tumor cells via the Fc fragment of rituximab. This study was designed to distinguish between these two possibilities. Since the CH2 domain in Fc is responsible for its interaction with the Fc receptor, we have genetically engineered a rituximab mAb in which the CH2 domain was deleted. The CHO cells secreted one monomeric form with properly formed disulfides rituximab (CH2−) and a dimeric form rituximab (CH2−) Dimer. The functions of the two CH2-deleted domain antibodies were examined in several systems and compared with those of the native rituximab. We used Ramos and 2F7 B-NHL cell lines as model systems. As expected, we demonstrate that the CH2-deleted antibodies are defective in their ability to mediate ADCC and CDC. However, their biological functions and their ability to signal the cells were not altered and were similar to those observed with rituximab. Hence, the CH2-deleted antibodies inhibited cell proliferation, induced cell aggregation in culture, and induced apoptosis when cross-linked with a secondary anti-human IgG. In addition, the CH2-deleted antibodies triggered the cells similar to rituximab and inhibited the Src kinase pLyn, the downstream p38 MAP kinase, the transcription factors NF-κB, YY1, SP1, and STAT3, and Bcl-2/Bcl-xL expression. These modifications resulted in the sensitization of B-NHL cell lines to both chemotherapeutic drugs and Fas-induced apoptosis. Altogether, these findings demonstrate that rituximab-mediated signaling and modification of intracellular signaling pathways can take place in the absence of any contribution of the Fc fragment cross-linked to the cell Fc receptors. These studies suggest that chemo and immuno-sensitization by rituximab can take place in vivo in the absence of Fc-FcR contributions and thus are of potential clinical relevance.

Description: Four patients from 3 Saudi Arabian families had delayed onset of immune deficiency due to homozygosity for a novel intronic mutation, g.31701T〉A, in the last splice acceptor site of the adenosine deaminase (ADA) gene. Aberrant splicing mutated the last 4 ADA amino acids and added a 43-residue “tail” that rendered the protein unstable. Mutant complementary DNA (cDNA) expressed inEscherichia coli yielded 1% of the ADA activity obtained with wild-type cDNA. The oldest patient, 16 years old at diagnosis, had greater residual immune function and less elevated erythrocyte deoxyadenosine nucleotides than his 4-year-old affected sister. His T cells and Epstein-Barr virus (EBV) B cell line had 75% of normal ADA activity and ADA protein of normal size. DNA from these cells and his whole blood possessed 2 mutant ADA alleles. Both carried g.31701T〉A, but one had acquired a deletion of the 11 adjacent base pair, g.31702-12, which suppressed aberrant splicing and excised an unusual purine-rich tract from the wild-type intron 11/exon 12 junction. During ADA replacement therapy, ADA activity in T cells and abundance of the “second-site” revertant allele decreased markedly. This finding raises an important issue relevant to stem cell gene therapy.

Description: Introduction: Asciminib is a new BCR-ABL1 inhibitor that differs from previous tyrosine kinase inhibitors (TKIs) in that it does not bind to the ATP-binding site of the kinase. Data from different clinical trials has shown an adequate safety and efficacy profile in chronic myeloid leukemia (CML) patients failing previous TKIs. However, no findings have been communicated in real life experience. The aim of our study is to present first results of asciminib in CML patients failing previous TKIs under the current compassionate use program. Methods: We retrospectively collected data from 31 patients treated with asciminib in 25 centers under compassionate use program. Data collecting was performed between October 2018 and June 2020. Patients baseline characteristics are shown in table 1. Most patients were heavily pretreated with 28 patients receiving 3 or more TKIs previous to asciminib. Eleven patients (35.5%) had been treated with ponatinib at some point throughout the disease. Twelve patients showed BCR-ABL1 mutations (only 1 case with T315I mutation). Switch to asciminib was due to intolerance in 22 patients and due to resistance in the remaining 9. Median dose of asciminib was 80mg per day (40mg every 12 hours). Treatment responses were evaluated according to European Leukemia Net recommendations. Data compilation and analysis were performed with REDCap Software and IBM SPSS (Version 25.0). Results: Median time on asciminib for the entire cohort was 35 weeks. Regarding toxicities, 13 patients (42%) experienced mild extra-hematological side effects (grade 1-2) being the most frequent fatigue (19%), joint pain (16%) and nausea (9%). Four patients (12,9%) showed severe (grade 3-4) extra-hematological events: fatigue, hepatotoxicity, hypertension and pericardial effusion (1 patient each). Three patients (9,7%) suffered from grade 4 thrombocytopenia, 2 of them associating grade 4 neutropenia. All toxicities according to previous TKIs adverse effects as well as cross-intolerance data is shown in table 2. Dose reduction had to be carried out in 9 patients (29%), 7 of those with temporary treatment interruptions; most owing to hematological adverse effects. In terms of efficacy (Graph 1), probability of reaching or at least maintaining previous response was 100%, 61.3% and 35.5% for complete hematological response (CHR), complete cytogenetic response (CCyR) and major molecular response (MMR), respectively. Regarding probabilities to improve previous responses, rates of CCyR and MMR were, respectively, 22,2% (2/9) and 22,2% (2/9) for resistant patients and 44% (4/9) and 62,5%. (10/16) for intolerant group. Amid the 11 patients previously treated with ponatinib, 3 patients (27,3%) showed improvement of response achieving at least MMR, 2 of them from the TKI-intolerant group and 1 from the TKI-resistant group. The median follow-up time was 40 weeks, after which 27 patients (87.1%) continued with asciminib. Treatment cessation happened in 2 patients due to progression to blastic phase and in 2 patients due to lack of efficacy. No patients discontinued due to side effects. Conclusion: The data presented, similar to that known from clinical trials, supports the use of asciminib in routine clinical practice in CML patients failing to previous TKIs. Disclosures Garcia-Gutiérrez: Novartis: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Bristol-Myers Squibb: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Pfizer: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Incyte: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding.