The effect of cellular nuclear function alteration on the pathogenesis of shoulder adhesive capsulitis: an immunohistochemical study on lamin A/C expression.

BACKGROUND: The network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamina. Lamins have been associated with important cellular functions: DNA replication, chromatin organization, differentiation of the cell, apoptosis and in maintenance of nuclear structure. Little is known regarding the etiopathogenesis of adhesive capsulitis (AC); recently, a dysregulating fibrotic response starting from a subpopulation has been described within the fibroblast compartment, which suddenly turns on an activated phenotype. Considering the key role of A-type lamins in the regulation of cellular stability and function, our aim was to compare the lamin A/C expression between patients with AC and healthy controls. MATERIALS AND METHODS: A case-control study was performed between January 2020 and December 2021. Tissue samples excised from the rotator interval were analysed for lamin A/C expression by immunohistochemistry. Patients with AC were arbitrarily distinguished according to the severity of shoulder flexion limitation: ≥ 90° and < 90°. Controls were represented by samples obtained by normal rotator interval excised from patients submitted to shoulder surgery. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed (Chi-square analysis; significance was set at alpha = 0.05). RESULTS: We enrolled 26 patients [12 male and 14 female, mean age (SD): 52.3 (6.08)] and 15 controls [6 male and 9 female, mean age (SD): 57.1 (5.3)]. The expression of lamin A/C was found to be significantly lower in the fibroblasts of patients with adhesive capsulitis when compared with controls (intensity of staining: p: 0.005; H-score: 0.034); no differences were found regarding the synoviocytes (p: > 0.05). Considering only patients with AC, lamin A/C intensity staining was found to be significantly higher in samples where acute inflammatory infiltrate was detected (p: 0.004). No significant changes in levels of lamin A/C expression were documented between the mild and severe adhesive capsulitis severity groups. CONCLUSIONS: Our study demonstrated that the activity of lamin A/C in maintaining nuclear structural integrity and cell viability is decreased in patients with adhesive capsulitis. The phase of the pathogenetic process (freezing and early frozen) is the key factor for cell functionality. On the contrary, the clinical severity of adhesive capsulitis plays a marginal role in nuclear stability. LEVEL OF EVIDENCE: III.

[Case Report: MPO-ANCA Associated Vasculitis After Pfizer-BioNTech COVID-19 mRNA Vaccination].

We report a case of MPO-anti-neutrophil cytoplasmic antibody ANCA-associated vasculitis, with pulmonary-renal syndrome, after the mRNA booster third dose vaccine Pfizer BioNTech against COVID-19 in 71-year-old Caucasian man with no specific past medical history. A kidney biopsy diagnosed ANCA-associated pauci-immune crescentic glomerulonephritis. Renal function and constitutional symptoms have been partially improved with treatment with dialysis, intravenous rituximab and steroid pulse therapy. No disease following either infection or vaccination with fourth dose against COVID-19.

MRI- and Histologic-Molecular-Based Radio-Genomics Nomogram for Preoperative Assessment of Risk Classes in Endometrial Cancer.

High- and low-risk endometrial carcinoma (EC) differ in whether or not a lymphadenectomy is performed. We aimed to develop MRI-based radio-genomic models able to preoperatively assess lymph-vascular space invasion (LVSI) and discriminate between low- and high-risk EC according to the ESMO-ESGO-ESTRO 2020 guidelines, which include molecular risk classification proposed by "ProMisE". This is a retrospective, multicentric study that included 64 women with EC who underwent 3T-MRI before a hysterectomy. Radiomics features were extracted from T2WI images and apparent diffusion coefficient maps (ADC) after manual segmentation of the gross tumor volume. We constructed a multiple logistic regression approach from the most relevant radiomic features to distinguish between low- and high-risk classes under the ESMO-ESGO-ESTRO 2020 guidelines. A similar approach was taken to assess LVSI. Model diagnostic performance was assessed via ROC curves, accuracy, sensitivity and specificity on training and test sets. The LVSI predictive model used a single feature from ADC as a predictor; the risk class model used two features as predictors from both ADC and T2WI. The low-risk predictive model showed an AUC of 0.74 with an accuracy, sensitivity, and specificity of 0.74, 0.76, 0.94; the LVSI model showed an AUC of 0.59 with an accuracy, sensitivity, and specificity of 0.60, 0.50, 0.61. MRI-based radio-genomic models are useful for preoperative EC risk stratification and may facilitate therapeutic management.

Nuclear lamin A in rotator cuff tear margin tenocytes: an antiapoptotic and cell mechanostat factor.

BACKGROUND: The network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamin. A- and B-type lamins are the major components of the nuclear lamina. Lamins function in many nuclear activities. The role of lamin A and transcription factors (NF-kB) as anti-apoptotic is well documented. Recently, lamin A has also been considered as a mechanosensor protein that is able to maintain nuclear integrity from mechanical insults. We aimed to verify how lamin A expression varies in healthy cuff cells and in those with different-sized tears where various mechanical stresses are present. METHODS: Forty-three patients with rotator cuff tear (RCT) [23M-20F, mean age (SD): 63.5 (6.1)] were enrolled. Tissue samples excised from the most medial point of tear margins were analyzed for lamin A expression by immunohistochemistry. Controls were represented by samples obtained by normal supraspinatus tendons excised from patients submitted to reverse shoulder prosthesis implant [8M-7F, mean age (SD): 67.9 (7.1)]. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed. RESULTS: Our study revealed a moderate intensity of lamin A in the healthy cuff tendons, a higher expression of this protein in the small tears, and a significant decrease of lamin A with increasing tear size (p < 0.0001). CONCLUSIONS: Our study emphasizes the importance of early repair of small RCTs since nuclear stability is maintained, and the cellular function is protected by lamin A overexpression. High re-tear of massive cuff repair could be due to cellular apoptosis and nuclear modifications induced by lamin A lack. LEVEL OF EVIDENCE: III.

PD-L1 expression is associated with tumor infiltrating lymphocytes that predict response to NACT in squamous cell cervical cancer.

Cancer immunotherapy has significantly improved the management of many malignancies in recent years. Although cervical cancer is the second most common women's cancer in the world, there are still few information about the role of checkpoint inhibitors in this neoplasm, especially in the neoadjuvant setting. In the present study, we retrieved 38 consecutive patients with squamous cell cervical cancer who underwent platinum-based neoadjuvant chemotherapy (NACT) followed by radical surgery. Pre-therapy biopsies were evaluated for the presence of tumor-infiltrating lymphocytes (TILs), including T (both cytotoxic CD8+ and helper CD4+) and B lymphocytes, macrophages, natural-killer cells, and eosinophils. Immunohistochemistry was performed to characterize the inflammatory cells and to evaluate programmed death-ligand 1 (PD-L1) expression on both neoplastic and inflammatory cells. We divided our study population in three groups using three cut-offs (< 10%, 10-40%, >40%), for both TILs and PD-L1 evaluation. Pathological response to NACT was obtained from the histological reports of the post-therapy surgical specimens. We observed that all cases showed stromal TILs, with a predominance of CD3+/CD4+ T helper cells, thus supporting the strong immunogenic potential of cervical cancer. The vast majority of neoplasms expressed PD-L1: 100% on immune cells and 92% on tumor cells. Firstly, we noticed that the percentage of neoplastic cells PD-L1+ was positively associated with high TIL percentage (p = 0.0073) and with increased PD-L1 expression on inflammatory cells (p = 0.0297). Secondly, we observed a significant correlation between both the percentage (p = 0.0105) of TILs and the expression of PD-L1 (p = 0.01045) on inflammatory cells and pathological response to NACT. These results suggest that cervical cancer could be a good target for immunotherapy, also in the neoadjuvant setting. Furthermore, PD-L1 expression was significantly associated with stromal TILs that interestingly may predict pathological response to NACT.

Targeting SMYD3 to Sensitize Homologous Recombination-Proficient Tumors to PARP-Mediated Synthetic Lethality.

SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors.

Antiarthritic Effects of a Root Extract from Harpagophytum procumbens DC: Novel Insights into the Molecular Mechanisms and Possible Bioactive Phytochemicals.

Harpagophytum procumbens (Burch.) DC. ex Meisn. is a traditional remedy for osteoarticular diseases, including osteoarthritis (OA), although the bioactive constituents and mechanisms involved are yet to be clarified. In the present study, an aqueous H. procumbens root extract (HPE; containing 1.2% harpagoside) was characterized for its effects on synoviocytes from OA patients and phytochemical composition in polyphenols, and volatile compounds were detected. HPE powder was dissolved in different solvents, including deionized water (HPEH2O), DMSO (HPEDMSO), 100% v/v ethanol (HPEEtOH100), and 50% v/v ethanol (HPEEtOH50). The highest polyphenol levels were found in HPEDMSO and HPEEtOH50, whereas different volatile compounds, mainly ß-caryophyllene and eugenol, were detected in all the extracts except for HPEH2O. HPEH2O and HPEDMSO were able to enhance CB2 receptor expression and to downregulate PI-PLC ß2 in synovial membranes; moreover, all the extracts inhibited FAAH activity. The present results highlight for the first time a multitarget modulation of the endocannabinoid system by HPE, likely ascribable to its hydrosoluble compounds, along with the presence of volatile compounds in H. procumbens root. Although hydrosoluble compounds seem to be mainly responsible for endocannabinoid modulation by HPE, a possible contribution of volatile compounds can be suggested, strengthening the hypothesis that the entire phytocomplex can contribute to the H. procumbens healing properties.

Phosphoinositide-specific phospholipase C isoforms are conveyed by osteosarcoma-derived extracellular vesicles.

Cancer cells are able to release high amounts of extracellular vesicles, thereby conditioning the normal cells in the surrounding tissue and/or in distant target organs. In the context of bone cancers, previous studies suggested that osteosarcoma cancer cells produce transforming extracellular vesicles able to induce a tumour-like phenotype in normal recipient cells. Indeed, phosphoinositide-specific phospholipase C (PI-PLC) enzymes are differentially expressed in osteosarcoma cell lines with increasing aggressiveness, thus providing helpful insights to better define their role and functions in this bone tumour. By confocal microscopy analysis, we demonstrated that osteosarcoma-derived extracellular vesicles convey all the assessed PI-PLC isoforms, and that they localize into cell membrane bubble-like structures, resembling extracellular vesicles about to be released, as conveyed and/or membrane protein. Cytofluorimetric analysis confirmed the presence of PI-PLC isoforms in the extracellular vesicles collected from conditioned media of osteosarcoma cells. These findings suggest the feasibility to use circulating extracellular vesicles as biomarkers of osteosarcoma progression and/or the monitoring of this distressing disease.

The Glucosamine-derivative NAPA Suppresses MAPK Activation and Restores Collagen Deposition in Human Diploid Fibroblasts Challenged with Environmental Levels of UVB.

The ultraviolet (UV) component of solar radiation is the driving force of life on earth, but it can cause photoaging and skin cancer. In this study, we investigated the effects of the glucosamine-derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-ß-D-glucose (NAPA) on human primary fibroblasts (FBs) stimulated in vitro with environmental levels of UVB radiation. FBs were irradiated with 0.04 J cm-2 UVB dose, which resulted a mild dosage as shown by the cell viability and ROS production measurement. This environmental UVB dose induced activation of MAP kinase ERK 1/2, the stimulation of c-fos and at lower extent of c-jun, and in turn AP-1-dependent up-regulation of pro-inflammatory factors IL-6 and IL-8 and suppression of collagen type I expression. On the contrary, 0.04 J cm-2 UVB dose was not able to stimulate metalloprotease production. NAPA treatment was able to suppress the up-regulation of IL-6 and IL-8 via the inhibition of MAP kinase ERK phosphorylation and the following AP-1 activation, and was able to attenuate the collagen type I down-regulation induced by the UVBs. Taken together, our results show that NAPA, considering its dual action on suppression of inflammation and stimulation of collagen type I production, represents an interesting candidate as a new photoprotective and photorepairing agents.

Phosphoinositide-specific phospholipase C in normal human liver and in alcohol abuse.

The phosphoinositide (PI) signal transduction pathway participates in liver metabolism. Abnormal activity or expression of PI-specific phospholipase C (PLC) enzymes has been described in different liver diseases. We resume the role of the PI metabolism in liver and PLC abnormalities in different liver diseases. Moreover, we present the results of PLC analyses in a normal human liver and an alcohol-damaged liver. PLC enzymes and the expression of the corresponding genes in liver biopsies from individuals deceased for complications of the alcoholic liver disease (ALD) at different stages compared with normal controls (deceased individuals with histologically normal livers without alcohol addiction anamnesis) were analyzed by using immunohistochemistry and molecular biology techniques. The expression panel of PLCs was described in normal and alcohol abuse liver. Our observations suggest that the regulation of PLC expression might be due to posttranscriptional events and that alcohol affects the epigenetic control of PLC expression belonging to PI signaling. We also describe the alternate expression of PLCB1 and PLCH1 genes in liver. Our results corroborate literature data suggesting that PLC enzymes are differently expressed in normal versus pathological liver, playing a role in the histopathogenesis of liver tissue damage. The expression and/or localization of selected PLC isoforms is especially affected in alcohol-related liver tissue histopathology. Our present observations confirm that the modulation of protein synthesis plays a role in the regulation of PLC enzymes. We also suggest that this modulation might act at the transcription level. Further studies are required to investigate related epigenetic mechanisms.

LPS, Oleuropein and Blueberry extracts affect the survival, morphology and Phosphoinositide signalling in stimulated human endothelial cells.

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes' expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC.

U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving Phosphoinositide-specific Phospholipases C.

The definition of the number and nature of the signal transduction pathways involved in the pathogenesis and the identification of the molecules promoting metastasis spread might improve the knowledge of the natural history of osteosarcoma, also allowing refine the prognosis and opening the way to novel therapeutic strategies. Phosphatydil inositol (4,5) bisphosphate (PIP2), belonging to the Phosphoinositide (PI) signal transduction pathway, was related to the regulation of ezrin, an ezrin-radixin-moesin protein involved in metastatic osteosarcoma spread. The levels of PIP2 are regulated by means of the PI-specific Phospholipase C (PLC) enzymes. Recent literature data suggested that in osteosarcoma the panel of expression of PLC isoforms varies in a complex and unclear manner and is related to ezrin, probably networking with Ras GTPases, such as RhoA and Rac1. We analyzed the expression and the subcellular localization of PLC enzymes in cultured human osteosarcoma MG-63 cells, commonly used as an experimental model for human osteoblasts, using U-73122 PLC inhibitor, U-73343 inactive analogue, and by silencing ezrin. The treatment with U-73122 significantly reduces the number of MG-63 viable cells and contemporarily modifies the expression and the subcellular localization of selected PLC isoforms. U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving PI-specific Phospholipases C.

Inter- and intra-patient clonal and subclonal heterogeneity of chronic lymphocytic leukaemia: evidences from circulating and lymph nodal compartments.

Whole exome sequencing and copy number aberration (CNA) analysis were performed on cells taken from peripheral blood (PB) and lymph nodes (LN) of patients with chronic lymphocytic leukaemia (CLL). Of 64 non-silent somatic mutations, 54 (84·4%) were clonal in both compartments, 3 (4·7%) were PB-specific and 7 (10·9%) were LN-specific. Most of the LN- or PB-specific mutations were subclonal in the other corresponding compartment (variant frequency 0·5-5·3%). Of 41 CNAs, 27 (65·8%) were shared by both compartments and 7 (17·1%) were LN- or PB-specific. Overall, 6 of 9 cases (66·7%) showed genomic differences between the compartments. At subsequent relapse, Case 10, with 6 LN-specific lesions, and Case 100, with 6 LN-specific and 8 PB-specific lesions, showed, in the PB, the clonal expansion of LN-derived lesions with an adverse impact: SF3B1 mutation, BIRC3 deletion, del8(p23·3-p11·1), del9(p24·3-p13·1) and gain 2(p25·3-p14). CLL shows an intra-patient clonal heterogeneity according to the disease compartment, with both LN and PB-specific mutations/CNAs. The LN microenvironment might contribute to the clonal selection of unfavourable lesions, as LN-derived mutations/CNAs can appear in the PB at relapse.

Increased chronic lymphocytic leukemia proliferation upon IgM stimulation is sustained by the upregulation of miR-132 and miR-212.

To assess the involvement of microRNAs (miRNAs) in B-cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross-linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR-132 and miR-212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR-132 and miR-212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR-132 and miR-212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR-132 and miR-212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention.

Minimal residual disease monitoring in chronic lymphocytic leukaemia patients. A comparative analysis of flow cytometry and ASO IgH RQ-PCR.

Minimal residual disease (MRD) is becoming increasingly important in chronic lymphocytic leukaemia (CLL) as treatment strategies are progressively improving. The primary objective of this study was to compare the applicability of three different flow cytometric approaches: basic 4-colour analysis, European Research Initiative in CLL (ERIC) consensus method and 8-colour analysis. Secondly, we investigated the sensitivity and specificity of flow cytometry (FC) compared to molecular analyses for MRD detection. A total of 462 CLL samples were evaluated by basic FC; in 143, ERIC consensus method was also performed and all three FC methodologies were applied in a subgroup of 10 cases. No discordance in defining MRD-positive/negative samples was observed between the FC methods; within positive samples, the ERIC consensus method and 8-colour analysis showed the most accurate results. MRD was analysed by FC and polymerase chain reaction (PCR) in 243 cases: concordant results were obtained in 199/243 samples (81·9%); 42/243 were FC-/PCR+. Overall, the sensitivity and specificity of FC compared to PCR was 96·5% and 77·2%, respectively. Both FC and PCR proved suitable for the detection of MRD and prediction of progression-free survival, which was significantly reduced in MRD-positive patients, regardless of the methodology. These results offer the rationale for a strategy to monitor MRD in CLL patients.

NOTCH1, SF3B1, BIRC3 and TP53 mutations in patients with chronic lymphocytic leukemia undergoing first-line treatment: correlation with biological parameters and response to treatment.

In chronic lymphocytic leukemia, NOTCH1, SF3B1, BIRC3 and TP53 disruptions are recurrent and affect survival. To define their incidence and clinical impact in patients undergoing first-line treatment, we evaluated 163 cases enrolled in the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) LLC0405 protocol (fludarabine plus alemtuzumab or fludarabine plus cyclophosphamide), for young patients, or in the ML21445 protocol (chlorambucil plus rituximab), for elderly patients. NOTCH1, SF3B1, BIRC3 and TP53 disruptions were detected in 15.9%, 12.2%, 8.6% and 10.4% of cases. NOTCH1 mutations correlated with a shorter treatment-free interval (p = 0.058), an unmutated immunoglobulin heavy variable gene (IGHV) status (p < 0.0001), CD38 and ZAP-70 expression (p = 0.0025 and 0.026, respectively) and trisomy 12 (p = 0.0028), SF3B1 mutations with an unmutated IGHV status (p = 0.02), and BIRC3 disruptions with an unmutated IGHV configuration (p = 0.01) and 11q deletion (p < 0.0001). NOTCH1 and SF3B1 did not appear to impact on overall response, while an inferior response was observed for BIRC3- and TP53-disrupted cases in the LLC0405 and ML21445 protocols, respectively. Progression-free survival, evaluable in the LLC0405 protocol - not affected by NOTCH1, SF3B1 and TP53 - appeared inferior for BIRC3 disruption. NOTCH1 and SF3B1 mutations may be overcome by aggressive regimens, while BIRC3 might impact on outcome also in intensive regimens.

IgD cross-linking induces gene expression profiling changes and enhances apoptosis in chronic lymphocytic leukemia cells.

Gene profile and functional changes upon IgD cross-linking were evaluated in chronic lymphocytic leukemia (CLL). Microarrays highlighted responsiveness to IgD in all cases, independently of clinico-biological characteristics. Stimulated samples exhibited the down-regulation of transcripts of B-cell receptor signaling and cell-adhesion at 24h and the up-modulation of differentiation and apoptosis genes at 48 h. A significant increase in apoptosis upon ligation was also documented. Furthermore, comparison between IgD and IgM stimulation displayed a differential transcriptional/functional response. In conclusion, CLL respond to IgD displaying expression changes and cell-death enhancement, indicating the apoptosis induction via-IgD as an alternative approach for CLL management.

Identification of molecular and functional patterns of p53 alterations in chronic lymphocytic leukemia patients in different phases of the disease.

We analyzed TP53 mutations in 483 chronic lymphocytic leukemia patients at different phases of the disease and found a higher incidence of mutations at the later phases and a distinctive mutation profile in each phase. p53 function evaluated by immunoblotting and flow cytometry after cell irradiation was impaired in 28 of 109 cases. Three phenotypically different dysfunctions were observed: type I, associated with heterozygous missense TP53 mutations (typically present at diagnosis) and partially resistant to radiation-induced killing; types II and III, with a higher incidence of microdeletions, nonsense mutations and bi-allelic TP53 defects (common in progressive and chemoresistant cases) and a complete radioresistance. Furthermore, in 4 of 28 patients, all chemoresistant, we found p53 dysfunctions without TP53 mutations. In chronic lymphocytic leukemia patients, a disease phase-specific variability in the p53 mutation profile and function takes place, and both analyses could be useful to guide treatment choices.

A subset of chronic lymphocytic leukemia patients display reduced levels of PARP1 expression coupled with a defective irradiation-induced apoptosis.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by defects in the DNA damage response and apoptosis. Among the factors involved in these pathways, we focused on the enzyme poly(ADP-ribose) polymerase 1 (PARP1) and on its substrate Che-1 by evaluating their basal expression and functional changes upon irradiation (IR). Microarray experiments were performed on 98 untreated CLL cases. Next, freshly isolated primary cells from 21 untreated patients were analyzed for in vitro response to irradiation through Western blot, PARP activity assay, Annexin-V analysis, and PARP1 basal expression by quantitative polymerase chain reaction. Microarray analysis showed that PARP1 and CHE1 were constitutively expressed in CLL and had a high degree of correlation with each other and with TP53. PARP1 and TP53 downmodulation was associated with worse clinical outcomes, especially in TP53-mutated cases. Next, CLL samples from 21 untreated patients were classified as responders and nonresponders based on IR-induced PARP1 cleavage. Notably, while responder samples were characterized by Che-1 and p53 induction at 8 hours and reduction at 24 hours post-IR, nonresponders included both samples with p53 dysfunctions and cases with a normal IR-induced Che-1 and/or p53 response. Finally, we observed that PARP1 was downregulated in nonresponder vs responder samples and that its basal expression was positively correlated with PARP1 cleavage after IR. In conclusion, we showed that reduced expression of PARP1 is associated with an impairment of CLL responsiveness to cell death.

White blood cell count at diagnosis and immunoglobulin variable region gene mutations are independent predictors of treatment-free survival in young patients with stage A chronic lymphocytic leukemia.

A comprehensive panel of clinical-biological parameters was prospectively evaluated at presentation in 112 patients with chronic lymphocytic leukemia (<65 years), to predict the risk of progression in early stage disease. Eighty-one percent were in Binet stage A, 19% in stages B/C. Treatment-free survival was evaluated as the time from diagnosis to first treatment, death or last follow up. In univariate analysis, advanced stage, hemoglobin, platelets, white blood cell, leukemic lymphocyte count, raised beta 2-microglobulin and LDH, unmutated immunoglobulin variable region genes, CD38, del(17p), del(11q) and +12, were significantly associated with a short treatment-free survival; the T/leukemic lymphocyte ratio was associated with a better outcome. Multivariate analysis of treatment-free survival in stage A patients selected a high white blood cell count and unmutated immunoglobulin variable region genes as unfavorable prognostic factors and a high T/leukemic lymphocyte ratio as a favorable one. At diagnosis, these parameters independently predict the risk of progression in stage A chronic lymphocytic leukemia patients.