In chronic lymphocytic leukemia (CLL), stabilizing mutations of NOTCH1, affecting up to 10-15% of cases, have been associated to poor prognosis, disease progression and refractoriness to chemotherapy. NOTCH1 mutations are significantly overrepresented in trisomy 12 CLL, a disease subset frequently expressing CD49d, the α4 chain of the very-late-activation-4 integrin, a well-known key regulator of microenviromental interactions, and negative prognosticator in CLL. In the present study, by analysing a wide cohort of 1180 CLL, we observed a very strong association between the presence of NOTCH1 mutations and the expression of CD49d (P<0.0001), occurring also outside the trisomy 12 CLL subset. Using both the MEC-1 CLL-like cells stably transfected with the NOTCH1 intracellular domain and primary CLL cells bearing a mutated or wild-type NOTCH1 gene configuration, we provide evidence that triggering of the NOTCH1 pathway resulted in a positive CD49d expression regulation, which was driven by a NOTCH1-dependent activation of nuclear factot-κB (NF-κB). Consistently, pharmacological inhibition of the NOTCH1 and/or of the NF-κB pathways resulted in impaired NF-κB nuclear translocation with consequent down-modulation of CD49d expression. Altogether, our data link for the first time NOTCH1 mutations to CD49d expression regulation through the involvement of the NF-κB pathway in CLL.
Ultrasound-guided alcoholization demonstrated a safe profile, relieved neuropathic symptoms in a majority of patients and improved their quality of life. Rescue therapy with surgery is feasible in patients with unsatisfactory response. However, a thorough evaluation for forefoot comorbidities should be obtained, as they may act as confounding factors.
Background and Objectives:Despite rarely, serous cystic adenoma (SCA) can assume a pseudo-solid aspect mimicking other pancreatic neoplasm as neuroendocrine tumor. EUS-FNA cytology has low diagnostic accuracy due to the scant cellularity of the collected samples. Histological diagnosis is usually made after resection. Recently, end-cutting needles for EUS-fine-needle biopsy (EUS-FNB), which obtain tissue cores by penetrating the lesions, have been developed. We aimed to assess the capability of EUS-FNB with SharkCore™ needles in the preoperative diagnosis of serous cystic adenoma pseudo-solid-appearing on imaging (Sa-SCA).Materials and Methods:Between January 2016 and January 2018, data from consecutive adult patients, who were referred for EUS-FNB of a solid pancreatic lesion and were diagnosed with having SCA, were retrieved from a single-center institutional database.Results:Two patients were excluded because of microcystic aspect at EUS. Histological diagnosis of SCA was made by EUS-FNB in the remaining 7 patients (5 females; mean age of 62.5 years). Lesions (mean size of 19.8 mm) were hypervascular on cross-sectional imaging, slightly hyperdense magnetic resonance imaging with T2-weighted images can, and negative at 68Ga-somatostatin receptor positron emission tomography and 18fluoro-deoxyglucose positron emission tomography. EUS-FNB samples were judged adequate for a definitive diagnosis in all cases, achieving specimens suitable for histological evaluation and several ancillary stains. Histochemical positivity for periodic acid-Schiff (PAS) and PAS with diastase digestion was observed in 7/7 cases. Immunohistochemical positivity for α-inhibin (7/7), GLUT1 (6/6), MUC6 (5/5), and negativity for synaptophysin (7/7) and chromogranin A (2/2) favored SCA diagnosis.Conclusions:In the case of preoperative workup suspected for Sa-SCA, a “forward acquiring” needle could improve the rate of preoperative histological diagnosis.
Background. The regulation of the B cell receptor (BCR)-mediated response to antigenic stimulation in chronic lymphocytic leukemia (CLL) is balanced by competing activating and inhibitory signals derived from specific co-receptors. In particular, the BCR inhibitory molecules Leukocyte associated Ig like receptor 1 (LAIR-1)/CD305 and human Fc receptor-like 2 (FCRL2)/CD307b have been independently reported as molecules associated with longer time to first treatment in CLL, although their synergic functional role and combined prognostic relevance remains to be explored. Aim. To assess the functional role and prognostic value of CD305 and CD307b as OS predictors in CLL. Methods. The study included 467 CLL cases all characterized at diagnosis for Rai stage (stages 0-I: 355 cases), CD49d expression (CD49d- CLL, <30% of positive cells by flow cytometry: 266), IGHV mutational status (mutated, M: 270), karyotype abnormalities according to the hierarchical stratification (normal/13q-/+12: 365). Median follow-up of patients was 70 months with 82 deaths. Immunophenotypic analysis was performed using a combination of anti-CD5 FITC, -CD19PE-Cy7, -CD305PerCPCy5.5, CD307bAPC mAbs. For functional assays, mouse anti-human IgM, and agonistic mouse anti-human CD305 and CD307b were used. Fab goat anti-mouse was used as cross linker. Phosphorylation state of ERK (pERK) and ATK (pATK) were evaluated by either flow cytometry or western blotting. Calcium flux was analysed by flow cytometry. Results.A significantly higher (p<0.0001) CD305 and CD307b expression in M versus UM CLL was documented, with mean % of expression of 58% vs. 33% (CD305), and 88% vs. 74% (CD307b). The best cut-off levels for OS, calculated using a ROC analysis, were 10% for CD305 and 85% for CD307b. Using these cut-offs, 331 (70.9%) and 296 (63.4%) were classified CD305+ and CD307bbright, respectively. The clinical impact of both markers as OS predictors was confirmed in both univariate (hazard ratio/confidence interval (HR/CI)= 0.39/0.25-0.60; p<0.0001 for CD305; HR/CI= 0.26/0.16-0.41; p<0.0001 for CD307b), and multivariate (HR/CI=0.41/0.26-0.63; p=0.0001 for CD305;HR/CI=0.27/0.17-0.42; p<0.0001 for CD307b) analyses. Therefore, we dichotomized CLL cases according to the expression of 2 markers (n=220) versus 0/1 markers (n=247). The prognostic impact of this combined markers expression was tested in univariate analysis (HR/CI=0.23/0.13-0.40; p<0.0001 ) and in a multivariate model including: IGHV mutational status, CD49d expression, Rai stage (stage 0-I versus stages II-IV), karyotype abnormalities (normal/del13/+12 versus del11/del17). The combined markers expression retained its prognostic impact (HR/CI=0.37/0.21-0.68; p=0.0013), along with the UM IGHV (HR/CI=2.54/1.51-4.26; p=0.0004), CD49d expression (HR/CI=1.83/1.13-2.97; p=0.015) del11/del17 (HR/CI=1.86/1.14-3.05; p=0.014). Consistently, expression of 2 markers identified subsets with longer OS in the context of both M (p=0.009) and UM CLL (p=0.004; see Figure). To functionally explain the peculiar clinical behavior of CLL expressing these molecules, we evaluated CLL cell response to BCR triggering in CD305+/CD307bbright CLL, with either a M (n=9) or a UM (n=7) IGHV gene status. A significant increase of anti-IgM response was found in 24-hour cultures compared to 2-hour cultures, irrespective of the IGHV mutational status: p-ERK, 41.2% vs 20% (p=0.006); pAKT, 18% vs 7% (p=0.037); calcium flux, mean AUC 2.0x106 vs 1.6x106 (p=0.01). This increased BCR response was paralleled by a significant down-regulation of both CD305 and CD307b after 24-hour culture compared to 2-hour culture (mean MFI 40 vs 92.5 for CD305, p=0.0009; 95.5 vs 414 for CD307b, p=0.001) with no difference between M and UM cases. We next tested the effects on BCR signaling of anti-CD305 and anti-CD307b ligation (n=4). While BCR engagement induced pERK (mean fold increase compared to the control=12), concomitant engagement of BCR and CD305 or CD307b reduced the pERK level to 68% or 40%, respectively. Moreover, simultaneous engagement of both CD305 and CD307b further inhibited pERK level to 28%. Conclusions.A CD305+/ CD307bbright phenotype predicts longer OS in CLL, in both M and UM IGHV CLL. The synergic effect of the B-cell receptor signaling inhibitors CD305 and CD307b may functionally explain this peculiar clinical behavior. Figure Figure. Disclosures Chiarenza: Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.
Background. Trisomy 12 (tris12) is a recurrent cytogenetic abnormality in chronic lymphocytic leukemia (CLL), occurring in approximately 15-20% of cases, often as the unique cytogenetic alteration, that is usually considered a clonal driver lesion occurring early in CLL evolution. In the Dohner hierarchical categorization, tris12 CLL are identified as having an intermediate prognostic risk, although recent reports suggest a more complex and heterogeneous clinical behavior. Compared to CLL lacking this cytogenetic abnormality, tris12 CLL show more atypical morphology and immunophenotype, more frequent expression of the negative prognostic markers CD49d and CD38, and presence of NOTCH1 mutations and an unmutated (UM) IGHV gene status. The increased fraction of tris12 CLL carrying adverse prognostic features is in contrast to the intermediate clinical behavior associated with most tris12 CLL cases. Aim. To perform a comprehensive evaluation of the clinical impact of the major genetic, immunogenetic and immunophenotypic prognostic markers in tris12 CLL. Methods. The study was based on a multicenter series of tris12 CLL defined according to Dohner (n=283, including 73 cases also bearing del13q), and a comparison group (control) of 553 cases with either del13q (n=308) or without any cytogenetic abnormality (no del17p, del11q, tris12, del13q, n=245). Median follow-up of patients in the tris12 and control groups were 4 years (range 0-22) and 7 years (range 0-28), with 54% and 57% treated patients, and 18% and 15% deaths, respectively. Patient characterization included modified Rai stage, CD49d (CD49dhigh, ≥30% positive cells by flow cytometry), CD38 (CD38high, ≥30% positive cells by flow cytometry) and ZAP-70 (ZAP-70high, ≥20% positive cells by flow cytometry) expression, and IGHV mutational status (mutated, M, or UM according to the 2% cutoff). TP53, BIRC3, NOTCH1 andSF3B1 mutations were screened either at diagnosis or before therapy by NGS with at least 1000X coverage and 1% of sensitivity. Groups were compared by chi-square test; overall survival (OS) was computed from diagnosis to death or censored at last observation, and analyzed by Cox regression analysis. Results. Comparing the tris12 and the control groups, median age was 64 years (range 30-92) vs 66 years (range 33-92), male gender 55% vs 56% (p=0.86), the modified Rai stage was early in 52% vs 54%, intermediate in 41% vs 42% and advanced in 7% vs 4% (p=0.20). As previously reported, tris12 CLL were characterized by a higher prevalence of cases expressing CD49d (85% vs 31%) and CD38 (62% vs 17%; all p<0.0001), and of UM IGHV cases (55% vs 25%, p<0.0001). Analysis of recurrent mutations highlighted a higher prevalence of NOTCH1 mutations (26% vs 8%, p<0.0001) and of BIRC3 mutations (21% vs 1%, p<0.0001) in tris12 vs control group CLL. Conversely, no differences were found in the fraction of cases with TP53 mutations (3% vs 4%, p=0.38) or SF3B1 mutations (7% vs 7%, p=0.89), and in cases expressing ZAP-70 (62% vs 52%, p=0.09). The impact of these features on OS was tested by univariate analysis: in tris12 CLL, only the UM IGHV gene status predicted shorter OS (HR=2.37, p=0.0063), while none of the other characteristics reaching statistical significance as OS predictors (CD49d HR=1.36, p=0.36; CD38 HR=0.42, p=0.052; ZAP-70 HR=3.12, p=0.07; TP53 HR=2.33, p=0.25; NOTCH1 HR=1.40, p=0.22; SF3B1 HR=2.05, p=0.17; BIRC3 HR=1.22, p=0.61). On the other hand, in the control cohort, a significantly higher HR was found for CD49d (HR 3.11, p<0.0001) and CD38 (HR 3.45, p<0.0001) expression, TP53 (HR 2.88, p=0.0026), NOTCH1 (HR 3.57, p<0.0001), and SF3B1 (HR 2.57, p=0.0038) mutations, as well as for the UM IGHV gene status (HR=2.81, p<0.0001), but not for ZAP-70 expression and BIRC3 mutations (HR=1.74 and HR=1.91, p=0.15 and p=0.37, respectively). Conclusions. Mutational status of IGHV genes was the sole prognostic factor able to stratify OS in tris12 CLL. Despite the high frequency of NOTCH1 and BIRC3 mutations, as well as of CD49d and CD38 overexpression, these markers failed to convey a prognostic risk in tris12 CLL. The lack of a significant clinical impact for TP53 and SF3B1 mutations might be partly explained by the low number of mutated cases combined with a relative short follow up in our tris12 cohort. These findings are in keeping with the hypothesis of a different patho-biological mechanism occurring in tris12 CLL, which however remains to be fully elucidated. Disclosures D'Arena: Janssen-Cilag: Honoraria. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Shanafelt:Genentech: Research Funding; Janssen: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.
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