Background Chimeric antigen receptor T (CAR-T) cells are an effective therapy in Diffuse large B-cell Lymphoma (DLBCL). However, more than half of patients (pts) still relapse. CAR-T cells from ...T-lymphocytes (T-Ly) enriched for early lineage T-cells have shown higher replicative potential and better efficacy. Recent studies have also shown that NK cells may enhance CAR-T cells antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CAR-T cells fitness. In the BioCART BS study, we assessed the T-cell subpopulations and NK cells at Ly-apheresis evaluating the impact of previous treatments. Methods Since April 2021, 27 DLBCL pts underwent Ly-apheresis at our Center. In Italy, until now, DLBCL pts are eligible to CAR-T cells therapy if relapsed/refractory after at least two lines of treatments. Since cryopreservation of the apheresed Ly is allowed for Tisa-cel, DLBCL pts with poor prognostic risk factor (primary refractory; PET positivity before Autologous Stem Cell Transplantation ASCT; relapse within 12 months) were enrolled in a “pre-emptive” Ly-apheresis program, scheduling leukapheresis as soon as possible. Combinations of monoclonal antibodies were used by Flow Cytometry to evaluate the CD4/CD8 ratios and T-cell subset percentages and counts. In particular of CD45RA+CCR7+ Naïve (N), CD45RA-CCR7+ Central Memory (CM), CD45RA-CCR7- Effector Memory (EM), and CD45RA+CCR7- Terminally Differentiated (TD) cells. NK cells were defined as CD3-CD56+/CD3-CD16+ cells. CAR-T cells expansion was evaluated using specific CD19 CAR reagent. All stained samples were acquired on a Canto II (BD Bioscience) flow cytometer and analyzed using DIVA software version 9.0. The CAR-T cells therapy response was evaluated at 30 days post infusion by CT or PET scan. Results 12 out of the 27 (44.4%) DLBCL pts underwent pre-emptive Ly-apheresis: 3 pts were primary refractory, 7 had PET+ before ASCT, while 2 relapsed within 1 year from the end of treatment program. Conversely, 15 pts were enrolled in the standard program, after at least two lines of treatment. The latter have been divided into 2 groups: pts who had received ASCT (n=9) and those who did not (n=6). At baseline, no clinical differences have been observed between the three groups. The median age was 61 years (29-73), while most pts had IPI >2 (68%) and Ann Arbor score>2 (82%). Comparing T-cell subpopulations at Ly-apheresis, pts who previously underwent ASCT presented a more “exhausted” T-Ly profile (Figure 1). Indeed, they displayed lower CD4/CD8 ratios compared with both the pre-emptive (p 0.03) and standard group without a history of ASCT (p 0.003). In addition, ASCT results in lower CD4+ Naïve T cells, both as percentage of parental cells and as absolute count, compared to the other two groups (p 0.003 and 0.005 for the pre-emptive group, p 0.005 for the group without previous ASCT). Conversely, pts who underwent ASCT had higher percentages and absolute counts of CD4+ EM T-Ly than pts in the pre-emptive group (p 0.003 and 0.05, respectively). They had also lower CD8+ Naïve T cells compared with those pts in the standard group who did not receive ASCT, while they displayed a trend towards increased absolute counts of highly differentiated CD8+ cells. Considering the NK-cells, pts who underwent ASCT had lower percentages of NK cells compared with pts of the pre-emptive group, as well as the standard group not receiving ASCT (p 0.01 and 0.002, respectively). This may reduce the CAR-T cells antitumor efficacy and CAR-T cell fitness. Of note, the T-Ly fitness impacts both on CAR-T cells expansion and efficacy. Indeed, pts who previously underwent ASCT less frequently had an adequate CAR-T cells expansion (“expander” pts: 25% vs. 75%) and a lower peak of CAR+ cells (4.5% vs. 21.9% CAR+ cells). In addition, pts who previously underwent ASCT displayed a lower, although not significant, complete remission rate at 30-days response compared with those pts who did not received it (20% vs. 44.4%). Notably, 5 out of 12 (42%) pts in the pre-emptive group had already activated a CAR-T cell program, showing its feasibility. Conclusion ASCT before Ly-apheresis results in more “exhausted” T-Ly profile and lower NK cells proportion at the time of leukapheresis. Timely pre-ASCT Ly-apheresis, as well as the use of CAR-T in second line would help to collect more “fit” Ly enriched with NK cells, and this may result in higher CAR-T cells expansion and efficacy.
•Bethlem myopathy (BM) may be inherited in a recessive manner.•Mutations may cause both Collagen VI reduction and impaired secretion and assembly.•The occurrence of recessive inherited BM implies ...changes in genetic counselling.
Bethlem myopathy represents the milder form of the spectrum of Collagen VI-related dystrophies, which are characterized by a clinical continuum between the two extremities, the Bethlem myopathy and the Ullrich congenital muscular dystrophy, and include less defined intermediate phenotypes. Bethlem myopathy is mainly an autosomal dominant disorder and the causing mutations occur in the COL6A genes encoding for the α1 (COL6A1), α2 (COL6A2) and α3 (COL6A3) chains. However, few cases of recessive inheritance have been also reported. We here describe clinical, genetic and functional findings in a recessive Bethlem myopathy family harbouring two novel pathogenic mutations in the COL6A2 gene. Two adult siblings presented with muscle weakness and wasting, elbows and Achilles tendon retractions, lumbar hyperlordosis, waddling gait and positive Gowers' sign. Muscle biopsy showed a dystrophic pattern. Molecular analysis of the COL6A2 gene revealed the novel paternally-inherited nonsense p.Gln889* mutation and the maternally-inherited p.Pro260_Lys261insProPro small insertion. Fibroblast studies in both affected patients showed the concomitant reduction in the amount of normal Collagen VI (p.Gln889*) and impairment of Collagen VI secretion and assembly (p.Pro260_Lys261insProPro). Each of the two variants behave as a recessive mutation as shown by the asymptomatic heterozygous parents, while their concomitant effects determined a relatively mild Bethlem myopathy phenotype. This study confirms the occurrence of recessive inherited Bethlem myopathy and expands the genetic heterogeneity of this group of muscle diseases.
Muscle pain may be part of many neuromuscular disorders including myopathies, peripheral neuropathies and lower motor neuron diseases. Although it has been reported also in mitochondrial diseases ...(MD), no extensive studies in this group of diseases have been performed so far. We reviewed clinical data from 1398 patients affected with mitochondrial diseases listed in the database of the “Nation-wide Italian Collaborative Network of Mitochondrial Diseases”, to assess muscle pain and its features. Muscle pain was present in 164 patients (11.7%). It was commonly observed in subjects with chronic progressive external ophthalmoplegia (cPEO) and with primary myopathy without cPEO, but also—although less frequently—in multisystem phenotypes such as MELAS, MERFF, Kearns Sayre syndrome, NARP, MNGIE and Leigh syndrome. Patients mainly complain of diffuse exercise-related muscle pain, but focal/multifocal and at rest myalgia were often also reported. Muscle pain was more commonly detected in patients with mitochondrial DNA mutations (67.8%) than with nuclear DNA changes (32.2%). Only 34% of the patients showed a good response to drug therapy. Interestingly, patients with nuclear DNA mutations tend to have a better therapeutic response than patients with mtDNA mutations. Muscle pain is present in a significant number of patients with MD, being one of the most common symptoms. Although patients with a myopathic phenotype are more prone to develop muscle pain, this is also observed in patients with a multi system involvement, representing an important and disabling symptom having poor response to current therapy.
Introduction
Patients with late-onset Pompe disease (LOPD) receiving enzyme replacement therapy (ERT) may develop IgG antibodies against alglucosidase alpha (anti-rhGAA) in the first 3 months of ...treatment. The exact role of these antibodies in modulating efficacy of ERT in this group of patients is still not fully understood. To assess whether anti rh-GAA antibodies interfere with ERT efficacy, we studied a large Italian cohort of LOPD patients.
Methods
We analyzed clinical findings and performed serial measurements of IgG anti rh-GAA antibody titers from 64 LOPD patients treated with ERT. The first examination (T0) was completed on average at 17.56 months after starting ERT, while the follow-up (T1) was collected on average at 38.5 months. Differences in T0–T1 delta of the six-minute walking test (6MWT), MRC sum score (MRC), gait, stairs and chair performance (GSGC) and forced vital capacity (FVC) were considered and then related to the antibody titers.
Results
Almost 22% of the patients never developed antibodies against GAA, while 78.1% had a positive titer (31.2% patients developed a low titer, 43.8% a medium titer and 3.1% a high titer). No statistical significance was found in relating the T0–T1 delta differences and antibody titers, except for MRC sum score values in a subgroup of patients treated < 36 months, in which those with a null antibody titer showed a greater clinical improvement than patients with a positive titer.
Conclusion
Our results confirm that in a large cohort of LOPD patients, anti rh-GAA antibody generation did not significantly affect either clinical outcome or ERT efficacy. However, in the first 36 months of treatment, a possible interference of low-medium antibody titers with the clinical status could be present. Therefore, a careful and regular evaluation of antibody titers, especially in cases with evidence of clinical decline despite ERT, should be performed.
Chronic progressive external ophthalmoplegia is a mitochondrial disorder usually caused by single or multiple mitochondrial DNA (mtDNA) deletions and, more rarely, by maternally inherited mtDNA point ...mutations, most frequently in tRNA genes (MTT). We report on a patient presenting with a progressive eyelid ptosis with bilateral ophthalmoparesis, dysphagia, dysphonia and mild proximal limb weakness associate with a mild movement disorder characterized by abnormal involuntary movements involving head and limbs, imbalance and gait instability. Muscle biopsy demonstrated the presence of ragged red fibers and several cytochrome-C-oxidase negative fibers. Molecular analysis showed the novel m.5613T > C heteroplasmic mutation in the mitochondrial tRNA(Ala) gene (MTTA) which disrupts a conserved site and fulfills the accepted criteria of pathogenicity. Moreover, a 38 CAG trinucleotide repeat expansion was found on the huntingtin gene, thus configuring a singular CPEO/"reduced penetrance" Huntington disease "double trouble". With this novel MTTA point mutation, we extend the spectrum of provisional pathogenic changes in this gene, which is a very rare site of pathogenic mutation, and confirm that clinical expression of these mutations is hardly ever heterogeneous, including myopathy and CPEO. Mitochondrial involvement is an emerging key determinant in the pathogenesis of Huntington disease and it is well known that mutant huntingtin influences the mitochondrial respiratory complexes II and III. A synergist effect of the HTT and MTTA mutations on respiratory chain function may be hypothesized in our patient and should be regarded as a spur for further studies on the mtDNA/HTT reciprocal interactions.
Chronic progressive external ophthalmoplegia is a mitochondrial disorder usually caused by single or multiple mitochondrial DNA (mtDNA) deletions and, more rarely, by maternally inherited mtDNA point ...mutations, most frequently in tRNA genes (MTT). We report on a patient presenting with a progressive eyelid ptosis with bilateral ophthalmoparesis, dysphagia, dysphonia and mild proximal limb weakness associate with a mild movement disorder characterized by abnormal involuntary movements involving head and limbs, imbalance and gait instability. Muscle biopsy demonstrated the presence of ragged red fibers and several cytochrome-C-oxidase negative fibers. Molecular analysis showed the novel m.5613T > C heteroplasmic mutation in the mitochondrial tRNA(Ala) gene (MTTA) which disrupts a conserved site and fulfills the accepted criteria of pathogenicity. Moreover, a 38 CAG trinucleotide repeat expansion was found on the huntingtin gene, thus configuring a singular CPEO/"reduced penetrance" Huntington disease "double trouble". With this novel MTTA point mutation, we extend the spectrum of provisional pathogenic changes in this gene, which is a very rare site of pathogenic mutation, and confirm that clinical expression of these mutations is hardly ever heterogeneous, including myopathy and CPEO. Mitochondrial involvement is an emerging key determinant in the pathogenesis of Huntington disease and it is well known that mutant huntingtin influences the mitochondrial respiratory complexes II and III. A synergist effect of the HTT and MTTA mutations on respiratory chain function may be hypothesized in our patient and should be regarded as a spur for further studies on the mtDNA/HTT reciprocal interactions.
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