Chronic pancreatitis is often secondary to alcohol abuse, but pancreatitis with no other aetiology is frequently associated with variants in genes encoding proteins related to zymogen granule ...activation. Our goal was to identify genomic variants in a patient by analyzing an extended panel of genes associated with the intra-pancreatic activation of the trypsin pathway. A 23-year-old woman was addressed at our institution because of chronic pancreatitis of unknown aetiology presenting recurrent episodes since she was the age of four. Next Generation Sequencing was performed to analyze a panel of nine genes associated with pancreatitis (CaSR, CFTR, CPA1, CTRC, CTSB, KRT8, PRSS1, PRSS2, and SPINK1). Three missense variants were found: p.Leu997Phe, maternally inherited, in the CFTR gene; p.Ile73Phe, paternally inherited, in the SPINK1 gene; and p.Phe790Ser, a de novo variant, in the CaSR gene. They were classified, respectively as probably benign, a Variant of Uncertain Significance, and the last one, which has never been described in the literature, as likely being pathogenic following American College of Medical Genetics and Genomics standard guidelines. Extensive intra-pancreatic activation of trypsin pathway gene sequencing detected rare variants that were not found with other gene screening and showed that variants in different genes may interact in contributing to the onset of the pancreatitis phenotype.
Inactivating
mutations causing Sotos syndrome have been previously associated with a specific genome-wide DNA methylation (DNAm) pattern. Sotos syndrome is characterized by phenotypic overlap with ...other overgrowth syndromes, and a definite diagnosis might not be easily reached due to the high prevalence of variants of unknown significance (VoUS) that are identified in patients with a suggestive phenotype.
we performed microarray DNAm profiling in a set of 11 individuals with a clinical suspicion of Sotos syndrome and carrying an
VoUS or previously unreported variants to solve uncertainty in defining pathogenicity of the observed variants. The impact of the training cohort size on sensitivity and prediction confidence of the classifier was assessed.
The Sotos syndrome-specific DNAm signature was validated in six individuals with a clinical diagnosis of Sotos syndrome and carrying
pathogenic
variants. Applying this approach to the remaining 11 individuals with
variants, we succeeded in confirming pathogenicity in eight subjects and excluding the diagnosis of Sotos syndrome in three. The sensitivity and prediction confidence of the classifier based on the different sizes of the training sets did not show substantial differences, though the overall performance was improved by using a data balancing strategy.
The present approach solved uncertainty in cases with
VoUS, further demonstrating the clinical utility of DNAm profiling.
▪
Background. Minimal residual disease (MRD) is the strongest prognostic factor in both children and adults with acute lymphoblastic leukemia (ALL). Currently, it is most widely monitored by ...molecular methods based on real-time-quantitative-PCR (RQ-PCR). Digital-droplet-PCR (ddPCR) and next-generation-sequencing (NGS) represent advanced tools that have the potential to overcome some limitations of standard approaches and potentially provide additional benefits. We analyzed adult ALL follow-up (FU) samples by RQ-PCR, ddPCR and NGS in order to better define the discriminating power of these novel methods.
Patients and Methods. Thirty adult ALL patients enrolled in the GIMEMA LAL 1913 protocol and their 83 FU bone marrow (BM) samples were studied. All patients received homogeneous induction/early consolidation chemotherapy, with concurrent MRD analysis at four time-points, to optimize risk classification and support risk/MRD-oriented therapy. RQ-PCR analyses followed the EuroMRD Consortium guidelines (van der Velden, 2007), ddPCR was performed as published (Della Starza, 2016; Cavalli, 2017) and NGS, as previously described (Faham, 2012; Kotrova M, 2017).
Results. By MRD RQ-PCR analysis, 19/83 samples were positive and quantifiable (Q), 9/83 were positive not-quantifiable (PNQ) and 55/83 were negative (NEG). By MRD ddPCR analysis, 27/83 samples were Q, 1/83 sample was PNQ and 55/83 proved NEG. Comparing the results of the two methods, we observed that MRD detection was concordantly positive or negative in 81% (67/83) of FU samples, while 19% (16/83) samples were classified as discordant. Most of the discordances occurred in samples with low levels of disease, i.e. PNQ or NEG: 9/83 were RQ-PCR PNQ, 4 of which were Q by ddPCR and 5 were ddPCR NEG. In the remaining 7 discordant FU samples, 5 were RQ-PCR NEG/ddPCR Q, 1 sample was RQ-PCR Q /ddPCR NEG and 1 sample was RQ-PCR NEG/ddPCR PNQ. The use of ddPCR significantly reduced the proportion of PNQ samples if compared to RQ-PCR - 1/83 (3%) vs 9/83 (15%) - respectively (p=0.0179), increasing the proportion of Q samples: 27/83 (33%) vs 19/83 (23%). It is worth noting that ddPCR also quantified the levels of disease in 9% (5/55) of samples, that were RQ-PCR NEG (Table 1).
MRD analysis was also performed by NGS in 41 samples from 15 patients: 18/41 samples proved Q and 23/41 were NEG. Comparing the MRD detection obtained by both ddPCR and NGS, we observed a concordant result in 98% (40/41) of samples; only 1 sample was ddPCR NEG and NGS Q with a MRD level of 1x10-5. The concordance between RQ-PCR and NGS was 78% (32/41 samples). Moreover, among these 41 samples 9 (from 7 patients) were discordant between RQ-PCR and ddPCR in the first comparative analysis: in 4 RQ-PCR-NEG FU samples, 3 were Q by both ddPCR and NGS, 1 was ddPCR NEG and NGS Q, with a MRD level of 10- 5; 1 subsequent relapse was observed; 4 FU samples that were RQ-PCR-PNQ/ddPCR-Q, were Q also by NGS; 1 subsequent relapse was observed. Finally, 1 RQ-PCR-PNQ sample was negative by both ddPCR and NGS, and no recurrence has so far been observed. Moreover, in the cohort of samples analyzed only by RQ-PCR and ddPCR, in 1 RQ-PCR NEG/ddPCR Q sample a relapse was observed, while the only case that was RQ-PCR Q/ddPCR NEG has so far not relapsed. Notably, 2 of the 3 relapses were documented in patients who were, at decisional treatment TPs, RQ-PCR PNQ or NEG and ddPCR/NGS Q.
Conclusions. When MRD levels are very low, it can be difficult to dissect if the not-quantifiable signal observed by PCR is due to few residual leukemic cells or to a non-specific amplification of normal DNA. The superior sensitivity and accuracy of ddPCR and NGS could be instrumental to univocally define these samples, which presently represent a problematic gray area in the clinical practice of MRD-driven protocols and might be associated with clinical relapse: indeed, among 83 FU samples analyzed we observed 3 relapses, whose FU samples were classified as PNQ or NEG by RQ-PCR, but proved Q by ddPCR and/or NGS. At variance, no relapses were recorded in patients whose FU samples were defined RQ-PCR-PNQ, but proved ddPCR/NGS NEG. Moreover, in 2/3 relapsed cases the change of MRD status (PNQ or NEG vs Q) could have led to a switch in risk classification and therefore in a treatment change. Further studies with a larger number of discrepant cases and a longer FU time will allow to conclusively define the clinical application and implication of these new methods.
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Chiaretti:Shire: Consultancy; Pfuzer: Consultancy; Amgen: Consultancy; Incyte: Consultancy. Foà:NOVARTIS: Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; INCYTE: Other: ADVISORY BOARD; AMGEN: Other: ADVISORY BOARD; GILEAD: Speakers Bureau.
Pancreatic cancer-melanoma syndrome (PCMS) is an inherited condition in which mutation carriers have an increased risk of malignant melanoma and/or pancreatic cancer. About 30% of PCMS cases carry ...mutations in CDKN2A. This gene encodes several protein isoforms, one of which, known as p16, regulates the cell-cycle by interacting with CDK4/CDK6 kinases and with several non-CDK proteins. Herein, we report on a novel CDKN2A germline in-frame deletion (c.52_57delACGGCC) found in an Italian family with PCMS. By segregation analysis, the c.52_57delACGGCC was proven to segregate in kindred with cutaneous melanoma (CM), in kindred with CM and pancreatic cancer, and in a single case presenting only with pancreatic cancer. In the literature, duplication mapping in the same genic region has been already reported at the germline level in several unrelated CM cases as a variant of unknown clinical significance. A computational approach for studying the effect of mutational changes over p16 protein structure showed that both the deletion and the duplication of the c.52_57 nucleotides result in protein misfolding and loss of interactors' binding. In conclusion, the present results argue that the quantitative alteration of nucleotides c.52_57 has a pathogenic role in p16 function and that the c.52_57delACGGCC is associated with PCMS.
Background: Inactivating NSD1 mutations causing Sotos syndrome have been previously associated with a specific genome-wide DNA methylation (DNAm) pattern. Sotos syndrome is characterized by ...phenotypic overlap with other overgrowth syndromes, and a definite diagnosis might not be easily reached due to the high prevalence of variants of unknown significance (VoUS) that are identified in patients with a suggestive phenotype. Objective: we performed microarray DNAm profiling in a set of 11 individuals with a clinical suspicion of Sotos syndrome and carrying an NSD1 VoUS or previously unreported variants to solve uncertainty in defining pathogenicity of the observed variants. The impact of the training cohort size on sensitivity and prediction confidence of the classifier was assessed. Results: The Sotos syndrome-specific DNAm signature was validated in six individuals with a clinical diagnosis of Sotos syndrome and carrying bona fide pathogenic NSD1 variants. Applying this approach to the remaining 11 individuals with NSD1 variants, we succeeded in confirming pathogenicity in eight subjects and excluding the diagnosis of Sotos syndrome in three. The sensitivity and prediction confidence of the classifier based on the different sizes of the training sets did not show substantial differences, though the overall performance was improved by using a data balancing strategy. Conclusions: The present approach solved uncertainty in cases with NDS1 VoUS, further demonstrating the clinical utility of DNAm profiling.
The dipeptidyl peptidase 3 (Dpp3) is a ubiquitous zinc-dependent aminopeptidase, participating in the activation or degradation of signaling peptides and in the Keap1−Nrf2 antioxidant pathway. The ...absence of Dpp3 in the Dpp3 knockout mouse model causes increased osteoclast activity, altered osteogenic function, sustained oxidative stress in the bone tissue, and bone loss. We aimed to assess the association of Dpp3 activity with bone fragility in postmenopausal osteoporosis and the impact of denosumab on enzymatic activity. We conducted a two-phase study including 69 postmenopausal women with severe osteoporosis and 36 postmenopausal women without osteometabolic conditions, as controls (cross-sectional phase). Subjects with severe osteoporosis were assessed at baseline and 14 days after the first denosumab administration (prospective phase). The results showed significant reduction in serum Dpp3 activity (expressed as nmoles of formed product/mg proteins/min) in patients vs. controls (0.791 ± 0.232 vs. 1.195 ± 0.338; p < 0.001), and significant association with bone mass at the femoral neck (r = 0.28, p = 0.02) in patients prior to treatment. We found a negative correlation between C-terminal telopeptide (CTX) or N-terminal pro-peptide of type 1 procollagen (P1NP) levels and Dpp3 activity (respectively, r = −0.29, p = 0.012; and r = −0.2572, p = 0.033). Dpp3 activity did not change after denosumab injection. Our findings support a critical role played by Dpp3 in bone homeostasis as a potential bone protective factor. Additional clinical studies in larger cohorts might explore the implementation of Dpp3 assessment as a biomarker of bone health status.
Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches ...allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections.
In this paper, a two-dimensional numerical investigation on a prototypal solar chimney system integrated with an absorbing capacity wall in a south facade of a building is presented. The capacity ...wall is composed of a high absorbing plate and an assigned thickness of phase change material in metal foam. The chimney consists of a converging channel with one vertical absorbing wall and the glass plate inclined of 2°. The channel height inside the chimney is equal to 4.0 m, whereas the channel gap is at the inlet equal to 0.34 m and at the outlet it is 0.20 m. The thermal energy storage system is 4.0 m high. The numerical analysis was intended to evaluate the thermal and fluid dynamic behaviors of the solar chimney integrated with a latent thermal energy storage system. The investigation has shown that in all cases PCM has not fully melted during the day and the presence of aluminum foam inside the box attenuates the variation of temperatures during the day. The results show that the three different thickness of the thermal storage system present very similar fluid dynamic and thermal behaviors. For the analyzed configurations, the phase change material does not reach a total melting during the considered day.
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