New CACNA1A deletions are associated to migraine phenotypes.

BACKGROUND: Familial hemiplegic migraine type 1 (FHM1) is a form of migraine with aura caused by heterozygous mutations in 4 genes: CACNA1A, ATP1A2, SNC1A and PRRT2, but further heterogeneity is expected. Here have been described clinical and molecular features in patients suffering from migraine with Aura (MA), without (MO) and hemiplegic migraine attacks. Next Generation Sequencing by TruSeq Custom Amplicon for CACNA1A and ATP1A2 gene has been performed. All genetic variants have been confirmed by Sanger sequencing and all samples were also analyzed with MLPA assay for ATP1A2-CACNA1A genes to detect duplication or deletion. All MLPA data were verified by Real Time PCR. RESULTS: Sequencing analysis showed 3 point mutations, two novel variants and one already described in literature. Moreover, MLPA analysis showed 3 deletions in 9 sporadic hemiplegic migraine (18%), in 3 patients with non-hemiplegic migraine (4.1%) and in 3 patients affected by episodic ataxia (20%). Two sporadic patients showed a deletion in exons 41-43, while the rest of HM patients (5) showed a deletion in the terminal part of the CACNA1A gene. About episodic ataxia, we have identified deletions in exon 12-15 and in exon 47. Finally, in migraine patients, we have found different subjects affected by different phenotypes deleted in exon 47. CONCLUSION: This work highlights the importance to complement analysis as direct sequencing with quantitative analysis (MLPA). In fact, intragenic CACNA1A rearrangements have been detected. Our work demonstrated that deletions in CACNA1A gene may be associated also to different migraine phenotypes.

Telomere length identifies two different prognostic subgroups among VH-unmutated B-cell chronic lymphocytic leukemia patients.

Some evidences suggest that telomere restriction fragment length (TRF-L) is an effective indicator of histopathogenesis in B-cell tumors. As histopathogenesis is relevant for B-cell chronic lymphocytic leukemia (B-CLL) prognosis, TRF-L was assessed by Southern blot in 201 patients and compared to variable immunoglobulin heave chain gene mutational status (VH-MS) and to other known prognostic features. Overall survival (OS), time to first treatment (TTFT) and progression-free survival (PFS) were evaluated. Our results indicate the following: (1) TRF-L is heterogeneous among B-CLL patients (median 6014 bp, range 1465-16 762); (2) TRF-L correlates to VH-MS (r(2)=0.1994, P<0.0001) with VH-mutated patients showing long and VH-unmutated short telomeres; however, 41% of VH-unmutated and 5% of VH-mutated patients did not show this correlation and were thus defined as 'discordant'; (3) TRF-L effectively predicts outcome in terms of TTFT, PFS and OS; (4) VH-unmutated discordant patients have a better clinical outcome than VH-unmutated concordant patients (OS P<0.01, PFS P<0.05) and similar to that of VH-mutated patients (OS, PFS P=NS). Compared to VH-unmutated concordant patients, VH-unmutated discordant patients showed no peculiarity in their immunoglobulin rearrangement nor in their flow cytometry or fluorescence in situ hybridization profile. In conclusion, TRF-L can be helpful to refine prognostication of B-CLL patients, particularly those with a VH-unmutated immunoglobulin sequence.

Marked telomere shortening in mobilized peripheral blood progenitor cells (PBPC) following two tightly spaced high-dose chemotherapy courses with G-CSF.

The purpose of the study was to compare telomere length (TL) in peripheral blood progenitor cells (PBPC) collected after two tightly spaced high-dose (hd) chemotherapy courses. We assessed 37 previously untreated lymphoma patients undergoing a hd-chemotherapy program with autografting. They sequentially received hd-cyclophosphamide (CY) and hd-Ara-C, both followed by PBPC harvesting. Both post-CY and post-Ara-C harvests were assessed for TL by Southern blot analysis. In 12 patients, the assay was also performed on purified CD34+ cells. All patients displayed high PBPC mobilization following both hd-CY and hd-Ara-C. In all but one patient, TL was shorter in PBPC collected after Ara-C compared to CY: 7226bp (range: 4135-9852) vs 8282 bp (range 4895-14860) (P < 0.0001). This result was confirmed on CD34+ cells. Platelet recovery in patients receiving post-Ara-C PBPC was significantly slower compared to those receiving post-CY PBPC. In conclusion, (i) administration of tightly spaced hd-chemotherapy courses induces marked telomere shortening on harvested PBPC; (ii) engraftment kinetics seem slower, with delayed platelet recovery, in patients autografted with PBPC suffering marked TL erosion; (iii) long-term follow-up is required to verify whether PBPC with shortened telomeres display defective engraftment stability and/or risk of secondary leukemia; (iv) TL evaluation is advisable whenever new mobilization procedures are developed.

Negative immunomagnetic ex vivo purging combined with high-dose chemotherapy with peripheral blood progenitor cell autograft in follicular lymphoma patients: evidence for long-term clinical and molecular remissions.

The feasibility and efficacy of a novel immunomagnetic ex vivo negative purging method was evaluated on peripheral blood progenitor cells (PBPC) from 13 non-Hodgkin's lymphoma patients (eight follicular, FL; three mantle cell, MCL; two FL with histologic transformation). A peculiar feature of the study was the collection of PBPC after prolonged tumor debulking. Our method included a stem cell enrichment phase followed by cell incubation with anti-B cell MoAbs (anti-CD19, CD20, CD22, CD23), addition of immunobeads, and then positive cell removal by passage on a Max-Sep (Baxter Immunotherapy) cell separator. Engraftment was rapid and stable. Hematological values were assessed 1 and 2 years after the autograft. Purging efficacy was molecularly assessed in a panel of 11 patients who showed persistence of PCR-detectable lymphoma cells on PBPC harvests despite intensified chemotherapeutic debulking. PCR-negativity was obtained in vitro and persisted in vivo after autograft in three FL patients; five more FL patients, whose purged PBPC were PCR+, converted to stable (3 patients) or fluctuating (two patients) PCR negativity after autograft. MCL patients never reached PCR negativity. Thus, ex vivo purging may have a role for FL patients harvesting PCR-positive PBPC after intensified chemotherapy. In contrast, the addition of ex vivo purging seems to be of little if any benefit for MCL patients.

Concurrent administration of high-dose chemotherapy and rituximab is a feasible and effective chemo/immunotherapy for patients with high-risk non-Hodgkin's lymphoma.

The aim of this study was to investigate feasibility, tolerability and efficacy of rituximab-supplemented high-dose sequential chemotherapy (R-HDS) with peripheral blood progenitor cell autografting as frontline or salvage treatment in patients with advanced non-Hodgkin's lymphoma (NHL). Thirty-two patients have been treated: 14 at disease onset and 18 with relapsed or progressive disease. R-HDS regimens included six courses of rituximab. Rituximab was delivered either concurrently with high-dose chemotherapy to exploit the in vivo purging properties of the drug as well as at the end of the treatment plan to target minimal residual disease. All patients treated at disease onset completed their treatment with no life-threatening toxicity, while two toxic deaths due to severe bilateral pneumonia were observed among patients treated due to relapsed or refractory disease. Thirteen of 14 patients treated up-front achieved CR. Among pre-treated patients 10 of 18 achieved CR with better results in patients with relapsed (seven of eight) compared to progressive disease (three of 10). PCR analysis was carried out in indolent lymphoma patients: nine of nine follicular lymphomas and three of six CD5-positive NHL collected PCR-negative peripheral blood progenitor cell harvests. The results of this pilot study show that R-HDS is feasible and effective with acceptable toxicity when used at disease onset. In pre-treated patients this treatment also showed promising results, although the risk of severe infections needs to be considered.

A validated real-time quantitative PCR approach shows a correlation between tumor burden and successful ex vivo purging in follicular lymphoma patients.

OBJECTIVE: Purging procedures are increasingly used to provide stem cell collections devoid of contaminating tumor cells. In follicle center lymphoma (FCL), most approaches eradicate polymerase chain reaction (PCR);-detectable disease in only a fraction of harvests undergoing ex vivo manipulation. In this study we evaluated whether there is a relationship between tumor burden of stem cell harvests and successful clearance of PCR-detectable disease following ex vivo manipulation. MATERIALS AND METHODS: To address this issue, we developed a real-time PCR approach for quantitative measurement of tumor contamination using the bcl-2 rearrangement. Real-time PCR was used to evaluate the relationship between tumor burden of stem-cell harvests and purging effectiveness in PCR(+) samples derived from 10 FCL patients. Ex vivo purging was performed using the MaxSep cell separator (Baxter Immunotherapy, Deerfield, IL, USA). RESULTS: Our real-time PCR method proved effective, sensitive, accurate, and reproducible. Four collections were successfully cleared of minimal residual disease (MRD) whereas six remained PCR(+). Real-time PCR showed that the four collections successfully cleared of MRD had a prepurging tumor burden significantly lower than those remaining PCR(+) (p = 0.04). CONCLUSION: This study provides the first evidence that evaluation of tumor burden in stem-cell harvests by real-time PCR can predict the effectiveness of therapeutic intervention in non-Hodgkin's lymphoma. Based on these findings, we foresee a more widespread use of this technique to evaluate the impact of different therapeutic approaches in FCL.

Central nervous system relapse in a patient with mantle cell lymphoma in continuous clinical and molecular remission at six years since autografting.

Although molecular remissions have been frequently observed and associated with low likelihood of relapse in some lymphoid tumours, they are seldom reported in mantle cell lymphoma (MCL). We performed PCR analysis of a MCL patient with central nervous system (CNS) relapse occurring 76 months after autologous transplantation. Molecular follow-up showed constant absence of PCR-detectable disease, even after the onset of relapse. These data indicate that isolated CNS relapse may occur even after several years of continuous remission and cannot be excluded based on a persistent pattern of molecular remission. However, the prolonged remission duration observed in this patient suggests that achieving PCR-negativity may also be of benefit for MCL patients.

Rituximab anti-CD20 monoclonal antibody induces marked but transient reductions of peripheral blood lymphocytes in chronic lymphocytic leukaemia patients.

Rituximab has been recently proposed as an effective non-chemotherapeutic option for patients with follicle centre lymphoma (FCL). However, less is known on its role in chronic lymphocytic leukaemia (CLL). We thus decided to assess its effectiveness on a panel of 7 patients with refractory or relapsed CLL. Mild (5 patients) or severe (1 patient) adverse reactions were observed during the first hours of Rituximab infusion, almost exclusively at the first course. Symptoms rapidly subsided with temporary drug withdrawal and low dose steroids. All patients could receive the whole scheduled treatment. A striking reduction of peripheral blood (PB) lymphocyte counts was observed in all patients (median 93%; range 57-99%). However, Rituximab was poorly effective towards nodal and splenic disease. Patients required additional treatment after a median time of 70 d (range: 20-180 d). Our data show that Rituximab delivery in CLL patients is feasible and has an acceptable toxicity, although it probably does not represent an ideal treatment option when delivered using schedules originally designed for FCL patients. However, responses observed at PB level suggest that Rituximab has an activity which is not negligible and deserves further investigation in CLL. Future approaches will be directed to the development of alternative schedules which may include dose intensification, combination of Rituximab and chemotherapy, and in vivo purging of peripheral blood progenitor cell harvests for autografting procedures.

Comparative assessment of telomere length before and after hematopoietic SCT: role of grafted cells in determining post-transplant telomere status.

Our objective was to characterize the role of grafted cells in determining telomere length (TL) after hematopoietic SCT (HSCT). A total of 20 patients undergoing autografts had PBSC collected after two sequential mobilization courses: TL in the first collection was significantly longer than in the second. For their autografts, 10 patients used PBSC from the first collection and 10 from the second. TL was also investigated before and after HSCT and on the graft in 10 allogeneic HSCT. After autografting, patients receiving PBSC from the first collection had BM TL reflecting that of grafted cells (median bp: 7730 on PBSC vs 7610 on post-HSCT BM, P=NS) and significantly longer than TL of the second collection; analogously, patients autografted with PBSC from the second collection had BM TL reflecting that of grafted cells (7360 on PBSC vs 7120 on post-HSCT BM, P=NS) and significantly shorter compared with the first collection. In the allograft setting, eight patients had their pre-transplant TL significantly shorter than donor PBSC (5960 vs 7110; P=0.0005); following HSCT, BM TL (median 7380 bp) was identical to that of the graft (P=NS). We conclude that grafted cells have a major role in determining TL after HSCT.

Telomere length is an independent predictor of survival, treatment requirement and Richter's syndrome transformation in chronic lymphocytic leukemia.

Telomere length (TL) has been associated with outcome in chronic lymphocytic leukemia (CLL). The aim of this extensive analysis carried out on 401 CLL patients was to assess TL conclusively as a prognostic biomarker. Our study included two cohorts used as learning (191 patients) and blinded validation series (210 patients). A TL cutoff of 5000 bp was chosen by receiver operating characteristic (ROC) analysis and Youden's index in the learning series. In this series, TL< or =5000 bp was independently associated to a worse outcome for both overall survival (OS; 105.5 vs 281 months, P<0.001) and treatment-free survival (TFS; 24.6 vs 73 months, P<0.001). In the blinded validation series, TL< or =5000 bp was confirmed as an independent outcome predictor for OS (79.8 vs not reached, P<0.001) and TFS (15.2 vs 130.8 months, P<0.001). Moreover, TL< or =5000 bp independently predicted the risk of Richter's syndrome (5-year risk: 18.9 vs 6.4%, P=0.016). Within CLL subsets defined by biological predictors, TL consistently identified patient subgroups harboring unfavorable prognosis. These results demonstrate that TL is a powerful independent predictor of multiple outcomes in CLL, and contributes to refine the prognostic assessment of this disease when utilized in combination with other prognostic markers. We thus believe that this prognostic biomarker has the potential for a more widespread use in CLL.

Prognostic significance of blood coagulation tests in lung cancer.

Previous studies have shown that activation of coagulation has an impact on the clinical course of lung cancer. This study was carried out to assess the potential prognostic significance of platelet count (P), prothrombin time (PT), partial thromboplastin time (PTT), anti-thrombin III (AT-III), fibrinogen (F), D-dimer (DD), factor II (F-II), factor VII (F-VII), factor X (F-X), protein C clotting (PCC), plasminogen (PL), and antiplasmin (AP) in 343 consecutive new lung cancer patients. A set of 32 anthropometric, clinical, physical, laboratory, radiological, and pathological variables was recorded prospectively for all patients. Patients were carefully followed-up, and their subsequent clinical course recorded. The most frequent abnormalities were of DD, F, and AT-III followed by F-VII, F-X, and F-II. Among the 12 clotting variables, the strongest relationships were those of F-II and F-X (Spearman rank (rs)=0.565), PT and F-VII (rs=0.562), F-VII and F-X (rs=0.514), PL and AP (rs=0.515), F and P (rs=0.490), AT-III and PCC (rs=0.476). Univariate analyses of survival showed that prolonged PT (p<0.043), and abnormally elevated DD (p<0.003), F (p<0.031), and P (p<0.047) were all associated with a poor prognosis. The multivariate model, however, did not confirm the prognostic significance of the coagulation factors. The results show subclinical activation of blood coagulation in lung cancer patients with early disease. In addition, clotting activation is confirmed as a predictor of survival, although not independently of other prognostic factors.