Annamaria Giordano

High Ki67 Index and Bulky Disease Remain Significant Adverse Prognostic Factors in Patients with Diffuse Large B Cell Lymphoma before and after the Introduction of Rituximab

The aim of this study was to evaluate the impact of clinical variables and biologic features on response rate (RR), overall survival (OS) and progression-free survival (PFS) in 111 patients with de novo diffuse large B cell lymphoma (DLBCL). Fifty-three patients were treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) and 58 patients were treated with R-CHOP (rituximab + CHOP). The variables predictive of RR in the CHOP group were B symptoms, age, clinical stage, bone marrow involvement, bulky disease, International Prognostic Index (IPI) and Bcl-2; in the R-CHOP group, these variables were bulky disease, bone marrow involvement, IPI and Ki67 expression >80%. Multivariate analysis showed that in patients treated with CHOP, the independent prognostic factors associated with PFS were age, bulky disease, IPI and Bcl-2 and those associated with OS were performance status, clinical stage, IPI and bone marrow involvement. In contrast, in patients treated with R-CHOP, the variable shown by multivariate analysis to be an independent prognostic factor associated with PFS was bulky disease, whereas Ki67 expression >80% was associated with OS and PFS. Our data show that a high Ki67 expression and bulky disease could represent possible predictive factors of poor prognosis, which would help to identify a high-risk subgroup of newly diagnosed DLBCL.

Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1mut FA, was observed in the relapsed cases compared to the NOTCH1mut allelic fraction observed in patients in complete or partial remission (median FA 4.75% vs 0.43%, respectively, p = 0.007). Our study demonstrated a much higher incidence of NOTCH1mut in CLL than has previously been reported, and showed that the NOTCH1mut allelic burden evaluation by ddPCR might identify patients in need of a closer clinical follow-up during the “watch and wait” interval and after standard chemotherapy.

Primary intracranial dural B cell small lymphocytic lymphoma

Primary central nervous system non-Hodgkin lymphomas (PCNSNHL) are extranodal lymphomas arising within and confined to the central nervous system (CNS). A frequency of 1 – 3% of all nonHodgkin lymphomas and 2 – 4% of all brain neoplasms has been reported, with a particularly high incidence in the AIDS population, in transplant recipients and immunocompromised patients [1 – 3]. Meningeal involvement by malignant lymphomas is most often a manifestation of diffuse aggressive lymphomas [1 – 3] while primary dural lymphomas with no associated cerebral parenchyma lesions are rare diseases [4 – 22]. We describe a case of primary B cell small lymphocytic lymphoma (SLL) involving the dura of the frontoparietal and temporal regions, and include a review of the literature, performing a Medline search for cases of primary intracranial dural lymphoma [4 – 22]. A 44-year-old immunocompetent woman referred a 2-week history of headache, diplopia, blurring of vision, loss of concentration, and right eye exophthalmos. Cranial magnetic resonance imaging (MRI) disclosed a large right dural thickening frontal mass, which was hyperintense in T1 weighted images and hypointense in T2 sequences. The administration of Gadolinium diethylenetriamine pentaacetic acid (Gd DTPA) revealed a wide contrast enhanced area involving the meninges and initial invasion of the underlying brain on the right frontoparietal and sphenoidal regions [Figure 1(A)]. Ophthalmic examination confirmed right eye exophthalmos and diplopia; fundoscopic evaluation showed mild pupil pallor, but no evidence of edema. Lumbar puncture was performed: cerebrospinal fluid (CFS) did not show malignant cells; CSF biochemical evaluations were within normal range with a glucose level of 65 mg/dl (n.v. 40 – 70 mg/dl) and protein level of 40 mg/dl (n.v. 15 – 45 mg/dl); polymerase chain reaction studies on CSF samples for Varicella Zoster, Herpes Simplex, Epstein-Barr viruses were negative. The patient underwent a right frontoparietal craniotomy. Intraoperatively, a diffuse plaque-like mass firmly adherent to the dura and infiltrating the pia mater and arachnoid membrane was encountered. The lesion could not be excised, and biopsies were performed. Histological examination revealed a monomorphic population of small lymphocytes diffusely infiltrating the dura [Figure 2(A)]. Immunohistochemical analysis demonstrated CD20 [Figure 2(B)], CD5 [Figure 2(C)], and CD23 [Figure 2(D)] positivity, bcl2 protein expression, and lambda light chain restriction, suggesting a typical pattern of SLL. CT scans of the neck, chest, abdomen, and pelvis showed no evidence of disease. Routine laboratory tests were within normal range, including serum lactate dehydrogenase 92 U/l (n.v. 100 – 190 U/l) and b2 microglobulin 1 mg/ml (n.v. 0.8 – 2.0 mg/ml). Cytological and immunophenotypical analysis did not show bone marrow and peripheral blood involvement. Serum HIV, EBV, CMV tests were negative. The patient was treated according to the IELSG 20 protocol and treatment consisted of four cycles of high dose i.v. methotrexate

Outcome of Very Late Relapse in Patients with Hodgkin's Lymphomas

Recurrences of Hodgkins Lymphoma (HL) 5 years after the initial therapy are rare. The aim of this study is to report a single centre experience of the clinical characteristics, outcome, and toxicity of pts who experienced very late relapses, defined as relapses that occurred 5 or more years after the achievement of first complete remission. Of 532 consecutive pts with classical HL treated at our Institute from 1985 to 1999, 452 pts (85%) achieved a complete remission. Relapse occurred in 151 pts: 135 (29.8%) within 5 years and 16 over 5 years (3.5%, very late relapses). Very late relapses occurred after a median disease-free interval of 7 years (range: 5–18). Salvage treatment induced complete remission in 14 pts (87.5%). At a median of 4 years after therapy for very late relapse, 10 pts (63%) are still alive and free of disease and 6 (37%) died (1 from progressive HL, 1 from cardiac disease, 1 from thromboembolic disease, 1 from HCV reactivation, and 2 from bacterial infection). The probability of failure-free survival at 5 years was 75%. The majority of deaths are due to treatment-related complications. Therapy regimens for very late relapse HL are warranted to minimize complications.

Validation of a biological score to predict response in chronic lymphocytic leukemia patients treated front-line with bendamustine and rituximab

During the the last two decades several biological prognostic markers have been identified in chronic lymphocytic leukemia (CLL) [1]. Some, like the IGHV mutational status and TP53 disruption, are also predictive of response to chemoimmunotherapy [2–6]. Rossi et al. reported an observational retrospective analysis on 404 CLL patients treated front-line with fludarabine-cyclophosphamide-rituximab (FCR) [6]. Based on the IGHV mutational status and FISH cytogenetics, patients were stratified into low risk (mutated IGHV and no adverse FISH cytogenetics [del(17p), del(11q)]), intermediate risk (unmutated IGHV and/or del11q in the absence of del17p), and high risk (del17p independent of co-occurring del11q or unmutated IGHV). This simple biologically based prognostic score based on the combination of three widely utilized biomarkers allowed to stratify patients with a significantly different progression-free survival (PFS) and overall survival (OS) after FCR treatment. In addition, they also demonstrated that low-risk patients had a durable remissions after FCR, with a life expectancy overlapping that observed in the age-matched general population [6]. Similarly, Laurenti et al. recently published a retrospective study on 102 patients with CLL treated front-line with chlorambucil-rituximab [7]. This analysis also showed that the above-mentioned biological score could distinguish patients with a different PFS. A trend toward a better OS was also observed. With the aim of investigating whether this biological score could also segregate CLL patients treated with bendamustine-rituximab (BR), we performed a retrospective study on previously untreated CLL patients who received BR as primary therapy. The cohort included 418 patients with progressive CLL from 34 different hematology centers (29 Italian, 3 Israeli, 1 German, and 1 American) who received at least one dose of BR as front-line treatment during the period 2008–2014; 279 of the 418 patients have been included in a previous multicenter study which reported on the front-line treatment of CLL patients with BR outside of clinical trials [8]. Relative survival, defined as the ratio between the actuarial survival observed in the CLL cohort and the expected survival of the general population matched by sex, age, nationality, and calendar year of starting BR was calculated using the Ederer II method. Expected survival estimates were calculated according to Italian, Israel, German, and American life expectancy tables (Human Mortality Database; http://www. mortality.org/, accessed 18 September 2017). Observed and expected survivals were compared using the Fisher test. Data analysis was carried out by STATA 13.1 (by StataCorp 4905 Lakeway Drive College Station, TX 77845, USA) (further details are in the Supplemental Appendix). The main clinical characteristics of the 418 patients are summarized in Table 1. After a median follow-up of 25 months, 86 patients progressed and 56 died, accounting for an estimated 2-year PFS of 73.2% and a 2-year OS of 88.9%. These results are similar to data of a multicenter prospective phase II trial enrolling 117 CLL patients who received BR as first-line therapy. Fisher et al. recorded a 2-year event-free survival of about 70% and a 2-year OS of 90% [3]. In 285 of the 418 cases complete molecular data were available (Table 1 * Massimo Gentile massim.gentile@tiscali.it

Assessment of DNA damages in lymphocytes of agricultural workers exposed to pesticides by comet assay in a cross-sectional study

Abstract Purpose: To assess the predictive power of the comet assay in the context of occupational exposure to pesticides. Materials and methods: The recruited subjects completed a structured questionnaire and gave a blood sample. Exposure to pesticides was measured by means of an algorithm based on Dosemeci’s work (Agricultural Health Study). Approximately 50 images were analyzed for each sample via fluorescence microscopy. The extent of DNA damage was estimated by tail moment (TM) and is the product of tail DNA (%) and tail Length. Results: Crude significant risks (odds ratios, ORs) for values higher than the 75th percentile of TM were observed among the exposed subjects (score > 1). The frequency of some confounding factors (sex, age and smoking) was significantly higher among the exposed workers. A significant dose–effect relationship was observed between TM and exposure score. Significant high-risk estimates (ORs), adjusted by the studied confounding factors, among exposure to pesticides and TM, % tail DNA and tail length were confirmed using unconditional logistic regression models. Conclusions: The adjusted associations (ORs) between the comet parameters and exposure to pesticides were significant. The sensitivity of the comet test was low (41%), the specificity (89%) and the predictive positive value (0.77) were found acceptable.

Risk of lymphoma subtypes by occupational exposure in Southern Italy

BackgroundOccupational exposure is known to play a role in the aetiology of lymphomas. The aim of the present work was to explore the occupational risk of the major B-cell lymphoma subtypes using a case–control study design.MethodsFrom 2009 to 2014, we recruited 158 lymphoma cases and 76 controls in the provinces of Bari and Taranto (Apulia, Southern Italy). A retrospective assessment of occupational exposure based on complete work histories and the Carcinogen Exposure (CAREX) job-exposure matrix was performed.ResultsAfter adjusting for major confounding factors, farmers showed an increased risk of diffuse large B-cell lymphoma (DLBCL) [odds ratio (OR) = 10.9 (2.3–51.6)] and multiple myeloma (MM) [OR = 16.5 (1.4–195.7)]; exposure to the fungicide Captafol was significantly associated with risk of non-Hodgkin lymphoma (NHL) [OR = 2.6 (1.1–8.2)], particularly with the risk of DLBCL [OR = 5.3 (1.6–17.3)].ConclusionsAgricultural activity seems to be a risk factor for developing lymphoma subtypes, particularly DLBCL, in the provinces of Bari and Taranto (Apulia Region, Southern Italy). Exposure to the pesticides Captafol, Paraquat and Radon might be implicated.Trial registrationProtocol number UNIBA 2207WEJLZB_004 registered 22/09/2008.

Peripheral blood CD4/CD19 cell ratio is an independent prognostic factor in classical Hodgkin lymphoma

Abstract Classical Hodgkin lymphoma (cHL) is characterized by the presence of tumoral cells in a rich background of T and B cells, macrophages and other inflammatory cells. The contribution of these non-tumoral cells to the pathogenesis of HL is still poorly understood. In our study we evaluated the prognostic significance of peripheral blood B, T and natural killer (NK) cells at diagnosis in 118 immunocompetent patients with cHL treated at our institution between January 2006 and December 2010. Fifty-four (46%) were male and 64 (54%) female. Median age at diagnosis was 33 years (range 15–82), and 71 patients (60%) presented an advanced stage (IIB–IV), 54 (46%) had bulky disease and 55 (47%) presented B symptoms. At the end of treatment, 94 patients (80%) had a complete response (CR) and 24 (20%) had a partial response. After a median follow-up of 54 months, 18 patients (15%) had relapsed. The variables that had a negative impact on progression-free survival (PFS) at univariate analysis were advanced stage, bone marrow involvement, International Prognostic Score (IPS) ≥ 3, positive interim positron emission tomography (int-PET), NK cells < 200/μL, CD19 cells < 85/μL, CD3/CD19 ratio ≥ 13 and CD4/CD19 ratio ≥ 10. At multivariate analysis, advanced stage, positive int-PET and CD4/CD19 ratio ≥ 10 were independent prognostic factors of PFS. New biological markers could be predictive of the response to treatment and survival in cHL. A CD4/CD19 ratio ≥ 10 seems to be associated with a worse outcome.

Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.

Predictive value of the CLL-IPI in CLL patients receiving chemo-immunotherapy as first-line treatment

An international collaboration has led to the development of a comprehensive tool [CLL-IPI international prognostic index for CLL] for the predicting of overall survival (OS) in chronic lymphocytic leukemia (CLL).1 CLL-IPI was based on data collected from 3500 CLL patients and was based on the following parameters: TP53 deletion and/or mutation, IGHV mutational status, β2-microglobulin plasma levels, clinical stage, and age. CLL-IPI provides the means to stratify CLL patients in the daily clinical practice (Supplementary Table 1).1 Although validated for OS2-4 and time to first treatment (TTFT),5 the predictive value of CLL-IPI on progression-free survival (PFS) has until now only been demonstrated in a single study on patients treated with chlorambucil (CLB), as monotherapy, or in combination with obinutuzumab or rituximab, as a first-line approach (CLL11 study),6 and presented as a poster at the annual meeting of the American Society of Hematology (ASH) in 2016. This article is protected by copyright. All rights reserved.