Adjuvant everolimus in high-risk diffuse large B-cell lymphoma: final results from the PILLAR-2 randomized phase III trial.

Background: Patients with diffuse large B-cell lymphoma (DLBCL) with an International Prognostic Index (IPI) ≥3 are at higher risk for relapse after a complete response (CR) to first-line rituximab-based chemotherapy (R-chemo). Everolimus has single-agent activity in lymphoma. PILLAR-2 aimed to improve disease-free survival (DFS) with 1 year of adjuvant everolimus. Patients and methods: Patients with high-risk (IPI ≥3) DLBCL and a positron emission tomography/computed tomography-confirmed CR to first-line R-chemo were randomized to 1 year of everolimus 10 mg/day or placebo. The primary end point was DFS; secondary end points were overall survival, lymphoma-specific survival, and safety. Results: Between August 2009 and December 2013, 742 patients were randomized to everolimus (n = 372) or placebo (n = 370). Median follow-up was 50.4 months (range 24.0-76.9). Overall, 47% of patients were ≥65 years, 50% were male, and 42% had an IPI of 4 or 5. 48% and 67% completed everolimus and placebo, respectively. Primary reasons for everolimus discontinuation versus placebo were adverse events (AEs; 30% versus 12%) and relapsed disease (6% versus 13%). Everolimus did not significantly improve DFS compared with placebo (hazard ratio 0.92; 95% CI 0.69-1.22; P = 0.276). Two-year DFS rate was 77.8% (95% CI 72.7-82.1) with everolimus and 77.0% (95% CI 72.1-81.1) with placebo. Common grade 3/4 AEs with everolimus were neutropenia, stomatitis, and decreased CD4 lymphocytes. Conclusions: Adjuvant everolimus did not improve DFS in patients already in PET/CT-confirmed CR. Future approaches should incorporate targeted agents such as everolimus with R-CHOP rather than as adjuvant therapy after CR has been obtained. ClinicalTrials.gov: NCT00790036.

Antiviral treatment in patients with indolent B-cell lymphomas associated with HCV infection: a study of the Fondazione Italiana Linfomi.

BACKGROUND: Tumor regression after antiviral therapy (AT) is in favor of an etiological role of hepatitis C virus (HCV) in non-Hodgkin's B-cell lymphomas (NHL). PATIENTS AND METHODS: We carried out a cohort study of 704 consecutive HIV-negative, HCV-positive patients with indolent NHL diagnosed and treated from 1993 to 2009 in 39 centers of the Fondazione Italiana Linfomi; 134 patients were managed with AT for lymphoma control. RESULTS: For entire cohort, 5-year overall survival (OS) was 78% [95% confidence interval (CI): 74%-82%] and 5-year progression-free survival (PFS) was 48% (95% CI: 44%-53%). In multivariate analysis, the use of AT during the patients' life had positive impact on OS. Forty-four of the 100 patients treated with first-line AT achieved a complete remission (CR) and 33 a partial response (PR). HCV-RNA clearance was achieved in 80 patients and was related to lymphoma response. At a median follow-up of 3.6 years, 5-year PFS was 63% (95% CI: 50%-73%). CR + PR rate was 85% with AT as second-line treatment. CONCLUSION: AT produces HCV-RNA clearance and consequent tumor regression in most patients with HCV-related indolent NHL. AT used at any time is associated with improved OS. Consequently, AT can be considered an option for patients with indolent lymphomas who do not need immediate cytoreductive treatment.

Impact of dose-dense immunochemotherapy on prognosis of germinal center and non germinal center origin of diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma. Gene-expression profiling in DLCBL has brought insight into the biological heterogeneity of the disease. Two major subgroups have been identified: germinal center B (GCB) cell and non-germinal center (non-GCB). The aim of this study was to define retrospectively by immunohistochemistry the bcell origin of 69 patients treated with R-CHOP14 and to evaluate if dose-dense therapy could improve their clinical outcome. According to immunohistochemistry analysis 28 patients were derived from germinal center and 41 from non-germinal center. After a median period of observation of 46 months (range 3-101 months) the overall survival (OS) was 75% and progression-free survival (PFS) was 53% and no differences were observed according to cell origin. In conclusion, we can point out that intensification could enhance the efficacy of the R-CHOP regimen and improve overall survival in patients with non germinal lymphoma.

Rituximab and subcutaneous 2-chloro-2'-deoxyadenosine as therapy in untreated and relapsed Waldenström's macroglobulinemia.

With the aim to assess the efficacy of subcutaneous cladribine in combination with rituximab, 29 newly diagnosed/pretreated WM patients were enrolled in a multicenter phase II trial. Intended therapy consisted of rituximab on day 1 followed by s.c. cladribine 0.1 mg/kg for 5 consecutive days, administered monthly for 4 cycles. The expression of genes involved in cladribine metabolism was evaluated. With a median follow-up of 50 months the ORR rate observed was 89.6% without any difference between newly or pretreated patients (P=.522). The therapy was well tolerated; no major infections were observed, no patients developed transformation to high-grade NHL nor myelodysplasia. Low expression levels of hCNT1 correlated with the failure to achieve a CR (P=.024). The combination of rituximab/cladribine is safe and effective in WM patients requiring treatment. The pharmacogenomic analysis suggests that hCNT1 might be beneficial in predicting clinical response to such a combination treatment.

Clinical activity of bortezomib in relapsed/refractory MALT lymphomas: results of a phase II study of the International Extranodal Lymphoma Study Group (IELSG).

BACKGROUND: The nuclear factor-kappa B activation in mucosa-associated lymphoid tissue (MALT) lymphoma pathogenesis provided the rationale for the evaluation of bortezomib in this malignancy. PATIENTS AND METHODS: Thirty-two patients with relapsed/refractory MALT lymphoma were enrolled. Thirty-one patients received bortezomib 1.3 mg/m(2) i.v., on days 1, 4, 8, and 11, for up to six 21-day cycles. RESULTS: Median age was 63 years (range, 37-82 years). Median number of prior therapies was 2 (range, 1-4). Nine patients had Ann Arbor stage I, 7 patients had stage II, and 16 patients had stage IV. Primary lymphoma localization was the stomach in 14 patients; multiple extranodal sites were present in 10 patients. Among the 29 patients assessable for response, the overall response rate was 48% [95% confidence interval (CI) 29% to 67%], with 9 complete and 5 partial responses. Nine patients experienced stable disease and six had disease progression during therapy. The most relevant adverse events were fatigue, thrombocytopenia, neutropenia, and peripheral neuropathy. After a median follow-up of 24 months, the median duration of response was not reached yet. Five deaths were reported, in two patients due to disease progression. CONCLUSION: Bortezomib is active in relapsed MALT lymphomas. Further investigations to identify optimal bortezomib dose, schedule, and combination regimens are needed since the frequent detection of dose-limiting peripheral neuropathy.

Expression of the human concentrative nucleotide transporter 1 (hCNT1) gene correlates with clinical response in patients affected by Waldenström's Macroglobulinemia (WM) and small lymphocytic lymphoma (SLL) undergoing a combination treatment with 2-chloro-2'-deoxyadenosine (2-CdA) and Rituximab.

PURPOSE: Resistance to nucleoside analogues agents is likely to be multifactorial and could involve a number of mechanisms affecting drug penetration, metabolism and targeting. In vitro studies of resistant human cell lines have confirmed that human concentrative nucleoside transporter 1 (hCNT1)-deficient cells display resistance. EXPERIMENTAL DESIGN: We applied real-time PCR method to assess the mRNA expression of equilibrative and concentrative nucleoside transporter (hENT1, hCNT1), deoxycytidine and deoxyguanosine kinase (dCK, dGK), 5'-nucleotidase (5'-NT), ribonucleotide reductase catalytic and regulatory (RR1, RR2) subunits in bone marrow cells from 32 patients with Waldenström's Macroglobulinemia (WM) and small lymphocytic lymphoma (SLL) who received 2CdA-based chemotherapy. Responses to chemotherapy, were then correlated to the expression of these markers. RESULTS: All 32 patients enrolled expressed lower levels of hCNT1 as compared to healthy donors. In univariate analysis, lower expression level of hCNT1 (p=0.0021) and RR2 (p=0.02) correlated with response to chemotherapy. In particular, patients with low levels of hCNT1 achieved inferior clinical response. No significant correlation between these genes expression and age, stage of disease was found. This study suggests that nucleotidase expression levels can be used to identify subgroups of WM and SLL patients who will likely respond differently to a 2CdA-based therapy.

Low dose Velcade, thalidomide and dexamethasone (LD-VTD): an effective regimen for relapsed and refractory multiple myeloma patients.

From January 2004, R/R MM cases referred to the Institution received LD-VTD regimen. Patients, irrespective of age, PS and life expectancy, were enrolled in the study once they had a measurable disease. Planned therapy: Velcade 1.0 mg m(-2) i.v. twice weekly for 2 weeks of a 28-day cycle for up to 6 cycles, oral Dexamethasone 24 mg on the day of and the day following each Velcade dose and Thalidomide 100 mg each evening. DVT prophylaxis with warfarin to maintain international normalized ratio between 2.0-3.0 was planned in all patients. As of 1 June 2005, 18 were the treated patients: median age 63 years, median time from diagnosis 5.8 years, a median of 4 previous therapy lines. Seventeen were the valuable patients and 9 (53%) were the responders: 2 CR, 6 PR, 1 MR. Six were the stable disease and 2 the progressive ones. Median time to best response was 2 months. Toxicity was negligible. No case of DVT was recorded. Except for the first cycle, subsequent cycles were delivered on an outpatient basis. After a median follow-up of 11 months, 12 patients were alive and 5 died (3 disease progression, 1 heart failure, 1 intestinal bleeding). Thus, the LD-VTD regimen applied appears feasible and effective in elderly and heavily pre-treated R/R myeloma patients.

The risk of second malignant tumors and its consequences for the overall survival of Hodgkin's disease patients and for the choice of their treatment at presentation: analysis of a series of 1524 cases consecutively treated at the Florence University Hospital.

PURPOSE: To quantify the incidence of second malignant tumors (SMT) as a whole and that of second "solid" tumors (SST) and leukemia (L) in a large series of 1524 Hodgkin's disease (HD) patients (pts) treated at the Florence University Hospital (UFH); to define the clinical and therapeutic features possibly related with SMT occurrence; to evaluate the consequences of SMT for the overall survival of the series studied and for the choice of the treatment of HD at presentation. METHODS AND MATERIALS: From 1960 to 1991, 1524 pts with HD, Clinical Stage (CS) I--IV have been treated at the UFH. Overall treatment consisted of radiation alone (RT, 36%), chemotherapy alone (CHT, 21%), or both (RT + CHT, 43%). The cumulative probability (CP) of SMT, SST, and L was calculated for the whole series and for the different clinical and therapeutic subgroups, and the results compared with uni- and multivariate analysis ("internal" comparison, IC). Standardized incidence ratios (SIR) for different SMT types (estimated on the basis of gender, age, period specific incidence rates of the general population) have been also calculated ("external" comparison, EC). The impact of the SMT-related mortality on the survival of the entire series has been estimated. RESULTS: A 14.9% 20-year CP of SMT was registered, along with a SIR of 2.04 (95% confidence interval [CI]: 1.2--2.5). Both IC and EC showed a statistically significant relationship between L incidence and treatment with CHT, alone or in combination with RT. A significant excess of breast cancers has been observed in RT-treated patients with longer follow-up (SIR, 2.9); an excess of other common SST (lung, non-Hodgkin's lymphomas) is evident in pts treated with either RT, RT + CHT, or CHT. The actuarial long-term survival of the series would have been better of about 3%, in absence of the SMT mortality possibly due to HD treatment, which is almost equally divided between patients treated with RT alone, CHT alone, and RT + CHT. CONCLUSIONS: SMT represent an important late event in HD long-term survivors. The relationship between L and treatment with CHT seems to be the most clearly defined. The effect of SMT on the survival of the entire series, although not negligible, does not seem to justify by itself substantial alterations in the current standards for the treatment of HD at presentation.

Survival in patients with intermediate or high grade non-Hodgkin's lymphoma: meta-analysis of randomized studies comparing third generation regimens with CHOP.

In patients with intermediate or high grade non-Hodgkin lymphoma (NHL), third generation chemotherapy regimens have been introduced to improve survival in comparison with the standard CHOP regimen. However, most studies have found no difference between these two treatments. We conducted a meta-analysis to assess the effectiveness of third generation regimens as compared with CHOP. Our study included the randomized controlled trials published in English from 1970 to 1999. After a Medline search, 5 trials were found to meet our inclusion criteria. A total of 1982 patients, that were enrolled in these trials, were included in the survival meta-analysis. Our methodology retrieved patient-level information from all of these subjects; survival up to 9 years after randomization was compared between the two treatment options. The results of our meta-analysis showed that, in comparison with CHOP, third generation chemotherapy did not prolong survival at levels of statistical significance (chi-square by log-rank test = 1.44, P = 0.23). The relative death risk for third generation regimens vs. CHOP was 0.92 (95%CI: 0.80 to 1.06;P = 0.26). We conclude that, on the basis of our meta-analysis, third generation regimens do not confer any survival benefit to patients with intermediate or high grade NHL as compared with CHOP.

Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues.

Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red-green-blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.

Treatment of large cell lymphoma with the Fi2 regimen (doxorubicin, vincristine, cyclophosphamide, bleomycin and prednisone): 25-year experience.

The CHOP protocol is the reference treatment for large cell lymphomas, but several other schemes of different intensity have recently been studied with controversial clinical findings. We report here the results obtained at our institution with a CHOP-like regimen called Firenze 2 (Fi2), which is characterised by an original scheduling of chemotherapy administration. A total of 225 patients, who were diagnosed from 1974 to 1996, were included in this retrospective study. All patients received the Fi2 regimen as a first-line intervention. One-hundred and sixty-two (72%) achieved complete remission; the overall survival at 120 months was 51% with a disease-free survival of 67% (median follow-up = 78 months). The survival curve showed a stable plateau of 42% after 16 years, which remained stable for further 4 years. In a multivariate survival analysis, achievement of complete remission (p<0.001) and IPI index of 0 or 1 (p=0.05) were significantly associated with a better survival. Overall, the outcome of our patients was similar to that reported by others, but the distinguishing feature of our study is the very long follow-up of the patients. Our study confirms that first generation regimens are effective and can cure a substantial proportion of patients. The long-term results of our study are helpful to retrospectively identify high-risk patients whose prognosis is poor and who can be candidates for more aggressive schemes of chemotherapy.

Prognosis of follicular lymphoma: a predictive model based on a retrospective analysis of 987 cases. Intergruppo Italiano Linfomi.

Patients (n-987) with a histologically confirmed diagnosis of follicular lymphoma were studied with the aim of developing a prognostic model specifically devised for this type of lymphoma. We collected information on age, sex, Ann Arbor stage, number of extranodal disease sites, bone marrow (BM) involvement, bulky disease, B symptom criteria (fever, night sweats, and weight loss), performance status (PS), serum lactate dehydrogenase (LDH) level, serum albumin level, hemoglobin level, and erythrocyte sedimentation rate (ESR). In the training sample of 429 patients with complete data, multivariate analysis showed that age, sex, number of extranodal sites, B symptoms, serum LDH level, and ESR were factors predictive for overall survival. Using these 6 variables, a prognostic model was devised to identify 3 groups at different risk. The 5- and 10-year survival rate was 90% and 65% for patients at low risk, respectively; 75% and 54% for patients at intermediate risk; and 38% and 11% for those at high risk (log-rank test, 86.62; P <. 0001). The model was also predictive (P =.0001) in the validation sample of 265 patients with complete data only for the 6 variables used in the development of the model and even in the group of 210 patients from the validation sample uniformly treated with doxorubicin-containing regimens (P =.0001). The prognostic model appears to be very useful in identifying patients with follicular lymphoma at low, intermediate, or high risk.

Immunohistochemistry applied to the study of bone marrow Gaucher's cells: a case report.

Gaucher's disease is frequently associated with immunologic abnormalities, e.g. hypergammaglobulinemia, polyclonal gammopathy and benign monoclonal gammopathy. A patient with Gaucher's disease and a selective accumulation of IgM k in Gaucher's cells without serum monoclonal gammopathies is described. The selective accumulation is detected by immunohistochemistry analysis performed on cryostat bone marrow biopsies.

Induction of apoptosis by monosaccharide butyrate stable derivatives in chronic lymphocytic leukemia cells.

BACKGROUND AND OBJECTIVE: Different therapeutic approaches are needed to restore apoptotic mechanisms in CLL cells, as present ones are not successful. We assessed the apoptotic effects of stable butyrate derivatives on CLL lymphocytes: in these molecules a mannose molecule is bound as ester to one-five butyrate moieties, conferring pharmacological stability to the pro-drugs which are able to induce apoptosis in primary AML blasts. DESIGN AND METHODS: Peripheral blood samples obtained from 17 patients with typical B-CLL were cultured in the presence of 0.5-1mM D1 (O-n-butanoyl-2, 3-O-isopropylidene-a-D-mannofuranoside), F1 (1-O-n-butanoyl-2, 3-O-isopropylidene-D,L-xylitol) and G1 (1-O-n-butanoyl-D,L-xylitol) derivatives for 4 days and equimolar sodium butyrate as comparison. After culture, apoptosis was evaluated by cell morphology, cellular DNA content, pattern of DNA fragmentation, annexin V exposure on cell membrane, and cell cycle parameters. Bcl2, bax, and fas oncogene expression were also evaluated by the APAAP method. RESULTS: The addition to cell cultures of D1 or F1 or G1 butyrate monosaccharides as well as sodium butyrate 0.5 and 1 mM determined to different extents an increase in the percentage of apoptotic cells in all CLL samples, relatively to the method and butyrate molecule added in culture. Heterogeneity in CLL cell sensitivity to the three butyrates was observed. Up to 60-68% apoptotic bodies were present in treated cultures after exposure to D1 0.5-1 mM, 60-72% after F1 0.5-1 mM and 48-60% after G1 0.5-1 mM. Comparison of untreated versus treated cultures yielded important significance (p< 0.001). At DNA content analysis, analyzed by flow cytometry, apoptotic events were accounting for up to 70-77% of D1-treated and 68-74% of F1-treated CLL cells at 0.5 and 1 mM concentrations (p= 0. 0001, vs controls 0-39%), and for 72-81% of G1 (0.5-1 mM) treated cells (overall, p=0.005). Cell cycle parameters were not altered by addition of butyrates, but expression of Annexin V was greatly enhanced. In a limited number of CLL cases fas, bcl2/bax ratio was analyzed and found unmodified. INTERPRETATION AND CONCLUSIONS: Monosaccharide butyrate stable derivatives are potent inducers of primary CLL cell apoptosis, both in untreated and alkylating agent pre-treated cases. Our results suggest that the apoptotic pathways elicited by butyrate in CLL lymphocytes are direct, specific and most probably do not involve bcl2/bax. Pro-apoptotic agents like the stable monosaccharide butyrate derivatives here studied could bring more insights into CLL biology and resistance to apoptosis, and possibly originate alternative treatments for CLL.

Regioselective synthesis and biological profiling of butyric and phenylalkylcarboxylic esters derivated from D-mannose and xylitol: influence of alkyl chain length on acute toxicity.

Regiospecific synthesis of 12 novel n-butyric and phenylalkylcarboxylic monoesters of mannose and xylitol was achieved. The strategy adopted, avoided a tedious intramolecular transesterification step, previously described for the synthesis of analogous compounds and permitted the facile synthesis of a new generation of stable derivatives. The general tolerance of the drugs has been assayed after intravenous administration of a bolus dose into mice. Monobutyric esters showed a low toxicity commensurate with the requirements for future development. A relationship was observed between chain length and toxicity. In contrast, phenylacetic, 3-phenylpropionic and 4-phenylbutyric esters were found to be toxic. Phenylbutyric esters induced marked and specific neuromuscular damage. Preliminary biological investigations of the new series of monobutyric esters showed them to retain the benificial biological properties of butyric acid whilst remaining relatively non toxic. They induced an inhibition of in vitro proliferation of 10 human cases of de novo acute myeloid leukemia (AML) primary cultures and AML established cell lines. AML blasts growth appeared to be blocked and cell differentiation was established. Transcription and expression of maturation markers and finally apoptosis were observed. Moreover, human gamma-chain hemoglobin (HbF) synthesis in erythroleukemia cells was stimulated by monobutyric esters. Mannose and xylitol butyric derivatives would appear to have exciting potential in treatment of beta-Hemoglobinopathies, sickle cell anemia and cancer.

Evaluation of breast tumour cell contamination in the bone marrow and leukapheresis collections by RT-PCR for cytokeratin-19 mRNA.

There is considerable interest in an autologous transplantation (AT) programme for patients with high-risk breast cancer; however, the issue of the incidence of occult bone marrow (BM) micrometastasis at diagnosis, and the cancer contamination of peripheral blood stem cell (PBSC) collections used for haematological rescue, is still debated. The presence of BM micrometastasis was evaluated in bilateral BM biopsies obtained at diagnosis of 33 patients with stage II/IIIA breast cancer using: (i) a 'nested' reverse transcriptase-polymerase chain reaction (RT-PCR) assay for cytokeratin 19 (K19) mRNA, (ii) histology, and (iii) immunohistochemistry (IHC) analysis with a panel of three monoclonal antibodies. The RT-PCR assay only was used to determine contamination of PBSC collections obtained after priming with recombinant human granulocyte-colony stimulating factor (rhG-CSF). K19 transcripts in one or both BM samples were detected in 48% of patients at diagnosis, with an overall 85% concordance with the results of IHC analysis. On the other hand, 56% of PCR- and IHC-positive BM samples were diagnosed as 'normal' on histological analysis. 57% of patients showed K19 mRNA in at least one PBSC collection; the possibility to have contaminated PBSC collections was significantly higher in patients with K19 positivity in BM at diagnosis. In four patients who had shown K19 positivity in BM and in PBSC collections, immunoselected CD34+ cells used for haematological rescue were K19-negative. There was a trend towards longer relapse free survival (RFS) in patients transplanted with K19-negative PBSC collections as compared to the others. In conclusion, a substantial proportion of patients with high-risk non-metastatic breast cancer present occult BM micrometastasis at diagnosis and also show cancer contamination of PBSC collections used for AT. These might represent a category of patients with poorer prognosis after AT, and possible candidates for more intensive and/or alternative therapeutic regimens, including AT with purged PBSCs.