The clonal evolution of two distinct T315I-positive BCR-ABL1 subclones in a Philadelphia-positive acute lymphoblastic leukemia failing multiple lines of therapy: a case report

BACKGROUND: The treatment of Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients who harbor the T315I BCR-ABL1 mutation or who have two or more mutations in the same BCR-ABL1 molecule is particularly challenging since first and second-generation Tyrosine Kinase Inhibitors (TKIs) are ineffective. Ponatinib, blinatumomab, chemotherapy and transplant are the currently available options in these cases. CASE PRESENTATION: We here report the case of a young Ph+ ALL patient who relapsed on front-line dasatinib therapy because of two independent T315I-positive subclones, resulting from different nucleotide substitutions -one of whom never reported previously- and where additional mutant clones outgrew and persisted despite ponatinib, transplant, blinatumomab and conventional chemotherapy. Deep Sequencing (DS) was used to dissect the complexity of BCR-ABL1 kinase domain (KD) mutation status and follow the kinetics of different mutant clones across the sequential therapeutic approaches. CONCLUSIONS: This case presents several peculiar and remarkable aspects: i) distinct clones may acquire the same amino acid substitution via different nucleotide changes; ii) the T315I mutation may arise also from an 'act' to 'atc' codon change; iii) the strategy of temporarily replacing TKI therapy with chemo or immunotherapy, in order to remove the selective pressure and deselect aggressive mutant clones, cannot always be expected to be effective; iv) BCR-ABL1-mutated sub-clones may persist at very low levels (undetectable even by Deep Sequencing) for long time and then outcompete BCR-ABL1-unmutated ones becoming dominant even in the absence of any TKI selective pressure

Initial Experience With Single-Isocenter Radiosurgery to Target Multiple Brain Metastases Using an Automated Treatment Planning Software: Clinical Outcomes and Optimal Target Volume Margins Strategy

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Determining Breast Implant Prevalence. A Population Study of Italian Chest Radiographs

Background Current breast implant prevalence within the general population remains elusive. An accurate prevalence is critical to serve as the denominator for any assessment of breast implant-related complication. The purpose of this manuscript is to assess this prevalence in women aged 20-70 years in Italy.Materials and Methods Eight reviewers, demonstrating a mean sensitivity of 87.0% and specificity of 97.0%, were recruited for retrospective identification of implants on chest radiographs from a tertiary academic hospital in a major urban setting. Three final reviewers were selected, and they assessed all eligible chest radiographs collected between January and December 2019. The hospital-based population was compared to epidemiological data at a local, regional and national level to demonstrate homogeneity of age structures using the phi correlation coefficient.Results We identified 3,448 chest X-rays which yielded 140 implants, with an overall prevalence of 4.1% for women aged 20-70. Implants were bilateral in 76% of cases and unilateral in 24%. They were placed cosmetically in 47.1% cases and used for reconstruction in 52.9% cases. Phi correlation coefficient found no differences across hospital-based, local, regional and national populations.Conclusion A validated method was performed to estimate implant prevalence from an academic hospital in a major urban setting at 4.1% and was used to estimate national prevalence in Italy. The implications of this epidemiologic study may reach across national borders for improved understanding of breast implant epidemiology and in predicting the total number of patients within a given population that may be affected by device complications

Serological and genetic evidence for altered complement system functionality in systemic lupus erythematosus: Findings of the GAPAID consortium

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of classswitched autoantibodies targeting nucleic acids

Use of a high sensitive nanofluidic array for the detection of rare copies of BCR-ABL1 transcript in patients with Philadelphia-positiveacute lymphoblastic leukemia in complete responseIlaria

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SNPs Array Karyotyping Reveals a Novel Recurrent 20p13 Amplification in Primary Myelofibrosis

The molecular pathogenesis of primary mielofibrosis (PMF) is still largely unknown. Recently, single-nucleotide polymorphism arrays (SNP-A) allowed for genome-wide profiling of copy-number alterations and acquired uniparental disomy (aUPD) at high-resolution. In this study we analyzed 20 PMF patients using the Genome-Wide Human SNP Array 6.0 in order to identify novel recurrent genomic abnormalities. We observed a complex karyotype in all cases, detecting all the previously reported lesions (del(5q), del(20q), del(13q), +8, aUPD at 9p24 and abnormalities on chromosome 1). In addition, we identified several novel cryptic lesions. In particular, we found a recurrent alteration involving cytoband 20p13 in 55% of patients. We defined a minimal affected region (MAR), an amplification of 9,911 base-pair (bp) overlapping the SIRPB1 gene locus. Noteworthy, by extending the analysis to the adjacent areas, the cytoband was overall affected in 95% of cases. Remarkably, these results were confirmed by real-time PCR and validated in silico in a large independent series of myeloproliferative diseases. Finally, by immunohistochemistry we found that SIRPB1 was over-expressed in the bone marrow of PMF patients carrying 20p13 amplification. In conclusion, we identified a novel highly recurrent genomic lesion in PMF patients, which definitely warrant further functional and clinical characterization

Italian real life experience with ibrutinib: Results of a large observational study on 77 relapsed/refractory mantle cell lymphoma

Although sometimes presenting as an indolent lymphoma, mantle cell lymphoma (MCL) is an aggressive disease, hardly curable with standard chemo-immunotherapy. Current approaches have greatly improved patients' outcomes, nevertheless the disease is still characterized by high relapse rates. Before approval by EMA, Italian patients with relapsed/refractory MCL were granted ibrutinib early access through a Named Patient Program (NPP). An observational, retrospective, multicenter study was conducted. Seventyseven heavily pretreated patients were enrolled. At the end of therapy there were 14 complete responses and 14 partial responses, leading to an overall response rate of 36.4%. At 40 months overall survival was 37.8% and progression free survival was 30%; disease free survival was 78.6% at 4 years: 11/14 patients are in continuous complete response with a median of 36 months of follow up. Hematological toxicities were manageable, and main extra-hematological toxicities were diarrhea (9.4%) and lung infections (9.0%). Overall, 4 (5.2%) atrial fibrillations and 3 (3.9%) hemorrhagic syndromes occurred. In conclusions, thrombocytopenia, diarrhea and lung infections are the relevant adverse events to be clinically focused on; regarding effectiveness, ibrutinib is confirmed to be a valid option for refractory/relapsed MCL also in a clinical setting mimicking the real world

Effects of inserting fluorescent proteins into the α1S II–III loop: insights into excitation–contraction coupling

In skeletal muscle, intermolecular communication between the 1,4-dihydropyridine receptor (DHPR) and RYR1 is bidirectional: orthograde coupling (skeletal excitation–contraction coupling) is observed as depolarization-induced Ca2+ release via RYR1, and retrograde coupling is manifested by increased L-type Ca2+ current via DHPR. A critical domain (residues 720–765) of the DHPR α1S II–III loop plays an important but poorly understood role in bidirectional coupling with RYR1. In this study, we examine the consequences of fluorescent protein insertion into different positions within the α1S II–III loop. In four constructs, a cyan fluorescent protein (CFP)–yellow fluorescent protein (YFP) tandem was introduced in place of residues 672–685 (the peptide A region). All four constructs supported efficient bidirectional coupling as determined by the measurement of L-type current and myoplasmic Ca2+ transients. In contrast, insertion of a CFP–YFP tandem within the N-terminal portion of the critical domain (between residues 726 and 727) abolished bidirectional signaling. Bidirectional coupling was partially preserved when only a single YFP was inserted between residues 726 and 727. However, insertion of YFP near the C-terminal boundary of the critical domain (between residues 760 and 761) or in the conserved C-terminal portion of the α1S II–III loop (between residues 785 and 786) eliminated bidirectional coupling. None of the fluorescent protein insertions, even those that interfered with signaling, significantly altered membrane expression or targeting. Thus, bidirectional signaling is ablated by insertions at two different sites in the C-terminal portion of the α1S II–III loop. Significantly, our results indicate that the conserved portion of the α1S II–III loop C terminal to the critical domain plays an important role in bidirectional coupling either by conveying conformational changes to the critical domain from other regions of the DHPR or by serving as a site of interaction with other junctional proteins such as RYR1