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Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix within the cardiac muscle, leading to increased stiffness and impaired heart function. From a rheological standpoint, knowledge about myocardial behavior is still lacking, partially due to a lack of appropriate techniques to investigate the rheology of in vitro cardiac tissue models. 3D multicellular cardiac spheroids are powerful and versatile platforms for modeling healthy and fibrotic cardiac tissue in vitro and studying how their mechanical properties are modulated. In this study, cardiac spheroids were created by co-culturing neonatal rat ventricular cardiomyocytes and fibroblasts in definite ratios using the hanging-drop method. The rheological characterization of such models was performed by Atomic Force Microscopy-based stress-relaxation measurements on the whole spheroid. After strain application, a viscoelastic bi-exponential relaxation was observed, characterized by a fast relaxation time (τ 1 ) followed by a slower one (τ 2 ). In particular, spheroids with higher fibroblasts density showed reduction for both relaxation times comparing to control, with a more pronounced decrement of τ 1 with respect to τ 2 . Such response was found compatible with the increased production of extracellular matrix within these spheroids, which recapitulates the main feature of the fibrosis pathophysiology. These results demonstrate how the rheological characteristics of cardiac tissue vary as a function of cellular composition and extracellular matrix, confirming the suitability of such system as an in vitro preclinical model of cardiac fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

The ability to measure mechanical response of cells under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Living cells have been mechanically investigated by several approaches. Among them, atomic force microscopy (AFM) is widely used thanks to its high versatility and sensitivity. In the case of large cells or 3D multicellular aggregates, standard AFM probes may not be appropriate to investigate the mechanical properties of the whole biological system. Owing to their size, standard AFM probes can compress only a single somatic cell or part of it. To fill this gap, we have designed and fabricated planar AFM macro-probes compatible with commercial AFM instruments. The probes are constituted of a large flat compression plate, connected to the chip by two flexible arms, whose mechanical characteristics are tuned for specific biological applications. As proof of concept, we have used the macro-probes to measure the viscoelasticity of large spherical biological systems, which have a diameter above 100 μm: human oocytes and 3D cell spheroids. Compression experiments are combined with visual inspection, using a side-view configuration imaging, which allows us to monitor the sample morphology during the compression and to correlate it with the viscoelastic parameters. Our measurements provide a quantitative estimate of the relaxation times of such biological systems, which are discussed in relation to data present in literature. The broad applicability of the AFM macro-probes can be relevant to study the biomechanical features in any biological process involving large soft materials. STATEMENT OF SIGNIFICANCE: The understanding of the role of physical factors in defining cell and tissue functions requires to develop new methods or improve the existing ones to accurately measure the biomechanical properties. AFM is a sensitive and versatile tool to measure the mechanical features from single proteins to single cells. When cells or cell aggregates exceed few tens of microns, AFM suffers from limitations. On these biological systems the control of the contact area and the application of a precise uniform compression becomes crucial. A modification of the standard cantilevers fabrication allowed us obtaining AFM macro-probes, having large planar contact area and spring constant suitable for biological investigations. They were demonstrated valuable to characterize the mechanical properties of large hierarchical biological systems., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: An increased incidence of second cancers has been reported in lymphoproliferative disorders., Patients and Methods: We assessed the frequency, characteristics and predictive factors of second cancers in 230 patients with Waldenström macroglobulinemia (WM) and compared the incidence of second cancers in WM with that of an age- and sex-matched control population., Results: Twenty-two patients (10%) developed solid cancers and 10 (4%) second hematologic malignancies. In a competing risk model, the cumulative incidence of solid cancers was 12% at 10 years and 17% at 15 years while the incidence of hematologic malignancies was 6% and 8%, respectively. The overall risk of second cancer in WM was 1.69 times higher than expected (P = 0.002). WM patients were at increased risk for diffuse large B-cell lymphoma [standardized incidence ratio (SIR) 9.24, P < 0.0001], myelodisplastic syndrome/acute myeloid leukemia (SIR 8.4, P < 0.0001), brain cancer (SIR 8.05, P = 0.0004). The risk of a second hematologic malignancy was fourfold higher in patients previously treated, though not reaching statistical significance (P = 0.19)., Conclusions: WM patients are at higher risk of second cancers as compared with the general population. The sample size does not allow firm conclusions about the effect of therapy on the development of second cancers.

Advanced age is one of the variables more frequently considered to be associated with an adverse prognosis in Waldenström's macroglobulinemia (WM). In a series of 238 symptomatic and asymptomatic WM patients, we retrospectively identified an age cut-off distinguishing two groups of patients with different outcome in terms of overall survival (OS), disease-specific survival (DSS) and treatment-free survival (TFS). Although for the OS the best cut-off was identified at 65 years with shorter OS for elderly patients, no difference was detected in terms of DSS between the two groups. Furthermore, patients over 65 years showed a longer TFS compared with patients under 65 years. Clinical and laboratory disease characteristics did not significantly differ between the two groups of patients except for β2M level. Therefore, the poorer survival of patients over 65 years at diagnosis should probably be attributed to the higher number of no disease-related deaths and is independent from WM.

Background: Hepatitis C virus (HCV) infection has been linked to lymphoproliferative disorders. Marginal zone B-cell lymphoma (MZL) represents one of the most frequent lymphoma subtypes associated with HCV infection. We describe an unusual subset of HCV-associated MZL characterized by subcutaneous presentation., Materials and Methods: A series of 12 HCV-positive patients presenting with subcutaneous nodules that revealed lymphoma infiltration at biopsy. Molecular analysis of immunoglobulin heavy chain (IGH) gene rearrangement and FISH investigations for t(11;18)(q21;q21) and t(14;18)(q32;q21) were carried out in nine patients., Results: The 12 patients (median age 69.5 years), all with positive HCV serology, presented with single or multiple subcutaneous nodules resembling lipomas. Histologically the lesions showed lymphoid infiltrates, consistent with extranodal MZL of mucosa-associated lymphoid tissue (MALT). Functional IGH gene rearrangements were identified in nine tested patients, with somatic mutations in 82%, indicating a histogenesis from germinal center-experienced B cells. The t(11;18) was found in two of nine cases. Staging did not show any other lymphoma localization. In two patients, a response was achieved with antiviral treatment. Extracutaneous spread to MALT sites occurred in a case., Conclusions: Our observations expand the spectrum of HCV-associated lymphomas to include a subset of extranodal MZL characterized by a novel primary 'lipoma-like' subcutaneous presentation and indolent clinical course.

Age older than 65 years, hemoglobin level lower than 100 g/L (10 g/dL), white blood cell count greater than 25 x 10(9)/L, peripheral blood blasts 1% or higher, and constitutional symptoms have been shown to predict poor survival in primary myelofibrosis (PMF) at diagnosis. To investigate whether the acquisition of these factors during follow-up predicts survival, we studied 525 PMF patients regularly followed. All 5 variables had a significant impact on survival when analyzed as time-dependent covariates in a multivariate Cox proportional hazard model and were included in 2 separate models, 1 for all patients (Dynamic International Prognostic Scoring System [DIPSS]) and 1 for patients younger than 65 years (age-adjusted DIPSS). Risk factors were assigned score values based on hazard ratios (HRs). Risk categories were low, intermediate-1, intermediate-2, and high in both models. Survival was estimated by the HR. When shifting to the next risk category, the HR was 4.13 for low risk, 4.61 for intermediate-1, and 2.54 for intermediate-2 according to DIPSS; 3.97 for low risk, 2.84 for intermediate-1, and 1.81 for intermediate-2 according to the age-adjusted DIPSS. The novelty of these models is the prognostic assessment of patients with PMF anytime during their clinical course, which may be useful for treatment decision-making.

Background: There are few data on the incidence and prognosis of extramedullary (EM) multiple myeloma (MM). There are concerns about a possible increase of EM relapses with the expanding use of high-dose therapy (HDT) and biological agents., Patients and Methods: The incidence of EM disease, its relationship with prior exposure to HDT or novel agents, and its prognostic impact were analyzed in 1003 MM patients. Based on the different therapies available, three periods were considered: 1971-1993, conventional-dose chemotherapy; 1994-1999, HDT for younger patients; and 2000-2007, introduction of novel agents., Results: Overall, 13% of patients had EM disease, 7% at diagnosis and 6% later. In the 2000-2007 period, there was a significant increase of EM involvement, at diagnosis (P = 0.02) and during follow-up (P = 0.03). The risk of EM spread was not significantly increased after HDT [hazard ratio (HR 0.6)], bortezomib (HR 1.62), or thalidomide/lenalidomide (HR 1.07). EM disease was associated with shorter overall (HR 3.26, P < 0.0001) and progression-free (HR 1.46, P = 0.04) survival., Conclusions: The incidence of EM disease has increased, probably due to the availability of more sensitive imaging techniques and the prolongation of patients' survival. HDT or novel agents seem not to increase the risk of EM disease. EM involvement confers a poor prognosis.

The conversion of the cellular form of the prion protein (PrP(C)) to an abnormal, alternatively folded isoform (PrP(Sc)) is the central event in prion diseases or transmissible spongiform encephalopathies. Recent studies have demonstrated de novo generation of murine prions from recombinant prion protein (recPrP) after inoculation into transgenic and wild-type mice. These so-called synthetic prions lead to novel prion diseases with unique neuropathological and biochemical features. Moreover, the use of recPrP in an amyloid seeding assay can specifically detect and amplify various strains of prions. We employed this assay in our experiments and analyzed in detail the morphology of aggregate structures produced under defined chemical constraints. Our results suggest that changes in the concentration of guanidine hydrochloride can lead to different kinetic traces in a typical thioflavin T(ThT) assay. Morphological and structural analysis of these aggregates by atomic force microscopy indicates a variation in the structure of the PrP molecular assemblies. In particular, ThT positive PrP aggregates produced from rec mouse PrP residues 89 to 230 lead to mostly oligomeric structures at low concentrations of guanidine hydrochloride, while more amyloidal structures were observed at higher concentrations of the denaturant. These findings highlight the presence of numerous and complex pathways in deciphering prion constraints for infectivity and toxicity.

Background: Among marginal zone lymphomas (MZLs), bone marrow (BM) involvement features are well established in the splenic marginal zone lymphoma (SMZL); few data are available for extranodal marginal zone lymphoma (EMZL) and nodal marginal zone lymphoma (NMZL)., Patients and Methods: Incidence and patterns of histologic BM involvement are studied in 120 MZL patients (48 SMZL, 59 EMZL, 13 NMZL) at onset and during follow-up; relationships between clinical features, BM histology and flow cytometry (FC) are analyzed., Results: At diagnosis, BM involvement occurs in 90% SMZL, 22% EMZL and 54% NMZL (P<0.0001); at reevaluation, incidence raises to 96% in SMZL and 34% in EMZL. Concordance between histology and FC is found in 87% of cases; most discordant cases have positive histology but negative FC. SMZL and EMZL show a nodular BM infiltration; the interstitial pattern is frequent in NMZL (P<0.0001); sinusoidal localization is typical of SMZL, frequent in NMZL and occasional in EMZL (P=0.0001). Stage, leukemic disease, B symptoms, more than one extranodal involved site, splenomegaly, elevated beta2-microglobulin, serum monoclonal component, International Prognostic Index (IPI) and age-adjusted IPI are directly related to BM infiltration., Conclusions: The different prevalence of BM involvement in MZL subtypes reflects their heterogeneous dissemination modalities; histology seems more sensible than FC to detect BM infiltration; development of BM involvement during follow-up is typical of EMZL.

Objective: Bortezomib is a new chemotherapeutic drug available for the treatment of lymphoid disorders, including multiple myeloma. Although its primary mechanism of action is proteasome inhibition, other mechanisms can contribute to the therapeutic effects, including modulation of inflammatory cytokines and immune response. One of the main toxic effects of bortezomib is peripheral neuropathy, usually occurring in the form of a painful, sensory axonal neuropathy. The mechanisms of peripheral damage, however, are still unclear. We here report a series of patients treated with bortezomib, who developed a peripheral damage possibly related to immuno-mediated, rather than toxic, mechanisms., Methods: Five patients who developed a peripheral neuropathy with severe motor involvement under bortezomib treatment underwent CSF, electrophysiological, and spinal cord MRI examinations., Results: Peripheral damage was characterized by: demyelinating or mixed axonal-demyelinating neuropathy, with prominent motor involvement; albumin-cytological dissociation; lumbar root enhancement on MRI in 2/5 patients; favourable outcome in 4/5 patients after immune treatments, either steroids (2 patients) or IVIg (2 patients)., Conclusions: In some instances, the peripheral damage associated with bortezomib may recognize immuno-mediated mechanisms., Significance: This form of bortezomib-associated neuropathy needs to be recognized as treatable condition, as it may respond to immune therapies. Unexplained worsening of neurological dysfunction despite bortezomib discontinuation, with prominent motor involvement and CSF signs of inflammation, may be the clues to this complication.

Background: To evaluate the clinical outcome of patients with relapsed or refractory follicular lymphoma treated with immunochemotherapy, in vivo purging and high-dose therapy with autotransplant., Patients and Methods: Sixty-four patients were enrolled in the trial. Primary end point was progression-free survival (PFS). Secondary end points were the in vivo purging effect on stem-cell harvest and the impact of molecular response on the outcome., Results: At enrollment, 59% of patients were PCR+ for bcl-2 rearrangement in bone marrow (PCR-informative). After the immunochemotherapy, before mobilization, 97% obtained complete response or partial response and 87% of patients informative for bcl-2 were molecularly negative. Sixty-one patients proceeded to in vivo purging and peripheral blood stem cell (PBSC) mobilization with rituximab and high-dose AraC. The median number of CD34+ cells collected was 16.6 x 10(6)/kg. Of 33 PCR-informative patients, the harvests resulted in PCR- in all. Fifty-eight patients received high-dose therapy and autotransplant of in vivo purged PBSC. After a median follow-up of 3.5 years, 41 patients are in complete remission. Five-year PFS is 59%., Conclusion: This study demonstrates that patients with advanced relapsed or refractory follicular lymphoma treated with immunochemotherapy, in vivo purging and autotransplant may obtain long-lasting PFS. In bcl-2-positive patients, in vivo purging allows the harvest of lymphoma-free PBSC. Absence of the bcl-2 rearrangement after autotransplant is associated with persistent clinical remission.

Post-polycythemia vera myelofibrosis (post-PV MF) is a late evolution of PV. In 647 patients with PV, we found that leukocytosis leukocyte count>(15x10(9)/L) at diagnosis is a risk factor for the evolution of post-PV MF. In a series of 68 patients who developed post-PV MF, median survival was 5.7 years. Hemoglobin level less than 100 g/L (10 g/dL) at diagnosis of post-PV MF was an independent risk factor for survival. The course of post-PV MF, however, is a dynamic process that implies a progressive worsening of clinical parameters. Using a multivariate Cox proportional hazard regression with time-dependent covariates, we found that a dynamic score based on hemoglobin level less than 100 g/L (10 g/dL), platelet count less than 100x10(9)/L, and leukocyte count more than 30x10(9)/L is useful to predict survival at any time from diagnosis of post-PV MF. The resulting hazard ratio of the score was 4.2 (95% CI: 2.4-7.7; P<.001), meaning a 4.2-fold worsening of survival for each risk factor acquired during follow up. In conclusion, leukocytosis at diagnosis of PV is a risk factor for evolution in post-PV MF. A dynamic score based on hemoglobin level, and platelet and leukocyte count predicts survival at any time from diagnosis of post-PV MF.

Essential thrombocythemia (ET) may occur in women of childbearing age. To investigate the risk of pregnancy complications, we studied 103 pregnancies that occurred in 62 women with ET. The 2-tailed Fisher exact test showed that pregnancy outcome was independent from that of a previous pregnancy. The rate of live birth was 64%, and 51% of pregnancies were uneventful. Maternal complications occurred in 9%, while fetal complications occurred in 40% of pregnancies. The Mantel-Haenszel method showed that fetal loss in women with ET was 3.4-fold higher (95% confidence interval [CI]: 3-3.9; P < .001) than in the general population. Half of the women studied carried the JAK2 (617V>F) mutation, and a multivariate logistic regression model identified this mutation as an independent predictor of pregnancy complications (P = .01). Neither the platelet count nor the leukocyte count was a risk factor. JAK2 (617V>F)-positive patients had an odds ratio of 2.02 (95% CI: 1.1 - 3.8) of developing complications in comparison with JAK2 (617V>F)-negative patients. Aspirin did not prevent complication in JAK2 (617V>F)-positive patients and appeared to worsen outcome in JAK2 (617V>F)-negative patients. A relationship was found between JAK2 (617V>F) and fetal loss (P = .05). This study indicates that patients carrying the JAK2 (617V>F) mutation have higher risk of developing pregnancy complications.

Background: Hepatitis C virus (HCV) infection is frequently associated with B-cell non-Hodgkin's lymphomas. We investigated the prevalence of HCV infection in nongastric marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) in order to define the relationship between the viral infection and the presenting features, treatment, and outcome., Methods: We retrospectively studied 172 patients with a histological diagnosis of marginal zone B-cell lymphoma of MALT, except for stomach, and with available HCV serology, among a series of 208 patients., Results: HCV infection was documented in 60 patients (35%). Most HCV-positive patients (97%) showed a single MALT organ involvement. HCV-positive patients showed a more frequent involvement of skin (35%), salivary glands (25%), and orbit (15%). The majority of stage IV HCV-positive patients (71%) had a single MALT site with bone marrow involvement. The overall response rate was similar in HCV-positive (93%) and HCV-negative patients (87%). Overall survival (OS) and event-free survival (EFS) did not differ according to HCV infection. In multivariate analysis, advanced disease (stage III-IV) was associated with a poorer OS (P = 0.0001), irrespective of HCV serostatus., Conclusions: This study shows that nongastric marginal zone lymphomas are characterized by a high prevalence of HCV infection. Patients with involvement of a single MALT site have the highest prevalence of HCV. HCV-positive nongastric lymphomas of MALT show an indolent course similar to HCV-negative patients and seem an ideal target for exploiting the antilymphoma activity of antiviral treatments.

Background: Diffuse large B-cell lymphoma (DLBCL) has been correlated to hepatitis C virus (HCV) infection in few series, but characteristics and outcome of these patients remain undefined., Patients and Methods: We analyzed 156 previously untreated consecutive HCV-positive patients with DLBCL observed between 1994 and 2004 in three major institutions from northern Italy., Results: Median age at presentation was 63 years and 8% of patients had DLBCL transformed from low-grade lymphomas. Spleen was the most frequently involved extranodal site, followed by liver and stomach. Treatment was delivered with cure-intent in 132 patients, while the remaining 24 patients received monochemotherapy or radiotherapy alone due to old age or seriously impaired hepatic function. Only five patients (4%) had to discontinue chemotherapy due to severe liver function impairment. The addition of rituximab did not seem to affect patients' tolerance to treatment. Five-year overall survival of the entire cohort was 72%, while 5-year progression-free survival (PFS) of the 132 patients treated with cure-intent was 51%. Hepatitis B virus co-infection, advanced Ann Arbor stage and nodal origin of the tumor resulted the strongest adverse prognostic factors., Conclusions: Patients with HCV-positive DLBCL share distinctive clinical features. Future studies should prospectively evaluate the association between HCV and aggressive lymphomas.

The Integruppo Italiano Linfomi (IIL) carried out a study to assess the outcomes of splenic marginal zone lymphoma and to identify prognostic factors in 309 patients. The 5-year cause-specific survival (CSS) rate was 76%. In univariate analysis, the parameters predictive of shorter CSS were hemoglobin levels below 12 g/dL (P < .001), albumin levels below 3.5 g/dL (P = .001), International Prognostic Index (IPI) scores of 2 to 3 (P < .001), lactate dehydrogenase (LDH) levels above normal (P < .001), age older than 60 years (P = .01), platelet counts below 100,000/microL (P = .04), HbsAg-positivity (P = .01), and no splenectomy at diagnosis (P = .006). Values that maintained a negative influence on CSS in multivariate analysis were hemoglobin level less than 12 g/dL, LDH level greater than normal, and albumin level less than 3.5 g/dL. Using these 3 variables, we grouped patients into 3 prognostic categories: low-risk group (41%) with no adverse factors, intermediate-risk group (34%) with one adverse factor, and high-risk group (25%) with 2 or 3 adverse factors. The 5-year CSS rate was 88% for the low-risk group, 73% for the intermediate-risk group, and 50% for the high-risk group. The cause-specific mortality rate (x 1000 person-years) was 20 for the low-risk group, 47 for the intermediate-risk group, and 174 for the high-risk group. This latter group accounted for 54% of all lymphoma-related deaths. In conclusion, with the use of readily available factors, this prognostic index may be an effective tool for evaluating the need for treatment and the intensity of therapy in an individual patient.

Distinct forms of tyrosine kinase domain (TKD), juxtamembrane domain, exon 8, and internal tandem duplication (ITD) mutations of c-KIT, were observed in about 46% of core binding factor leukemia (CBFL) patients. To evaluate their prognostic significance, 67 adult patients with CBFL were analyzed to ascertain the c-KIT mutation status. In acute myeloid leukemia (AML) with t(8;21), the presence of c-KIT TKD mutation at codon 816 (TKD(816)) was associated with a high white blood cell count at diagnosis (median, 29.60 x 10(9)/L) and a higher incidence (33%) of extramedullary leukemia (EML) during the course of the disease. Data also showed that the TKD(816) mutated patients (n = 12) had a significantly higher incidence of relapse and a lower overall survival (OS) at 24 months, compared with the 17 c-KIT unmutated (c-KIT(-)) patients (90% vs 35.3%, P = .002; 25% vs 76.5%, P = .006, respectively). No difference in relapse incidence (P = .126) and OS (P = .474) was observed between the c-KIT mutated other than TKD(816) (n = 7) and the c-KIT(-) patients. These findings indicate that c-KIT TKD(816) mutation has a negative impact on the outcome of AML with t(8;21).

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34(+) cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34(+) cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34(+) cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34(+) cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.

Shortly before the all-trans retinoic acid (ATRA) era, the GIMEMA cooperative group initiated a randomized study comparing idarubicin (IDA) alone with IDA plus arabinosylcytosine (Ara-C) as induction treatment in patients with newly diagnosed hypergranular acute promyelocytic leukemia (APL). Of the 257 patients evaluable for induction treatment, 131 were randomized to receive IDA alone (arm A) and 126 to receive IDA + Ara-C (arm B). Treatment in arm A consisted of 10 mg/m(2) IDA daily for 6 consecutive days, whereas in arm B it consisted of 12 mg/m(2) IDA daily for 4 days combined with 200 mg/m(2) Ara-C daily in continuous infusion for 7 days. Once in complete remission (CR), patients received 3 consolidation courses of standard chemotherapy, and those still in CR at the end of the consolidation were randomized to receive or not receive 1 mg/kg 6-mercaptopurine daily and intramuscular injections of 0.25 mg/kg methotrexate weekly for 2 years. Overall, 100 (76.3%) patients in arm A and 84 (66.6%) patients in arm B achieved CR (P = NS). Event-free survival (EFS) rates were 35% and 23% for patients in arm A and arm B, respectively (P =.0352). Multivariate analysis revealed that EFS was favorably influenced by induction treatment with IDA alone (P =.0352) and unfavorably influenced by white blood cell (WBC) counts greater than 3000/microL (P =.0001) and increasing age (P =.0251). These results indicate that anthracycline monochemotherapy with IDA favorably influences the EFS of patients with newly diagnosed hypergranular APL.

Minimal residual disease (MRD) following sequential administration of CHOP and rituximab was studied in previously untreated patients with follicular lymphoma. At diagnosis, the presence of Bcl-2/IgH-positive cells in the peripheral blood (PB) and/or bone marrow (BM) was demonstrated in all patients (n = 128) by polymerase chain reaction (PCR) analysis. Patients who achieved a clinical response following CHOP but remained PCR-positive were eligible for rituximab (375 mg/m(2) intravenously, weekly for 4 weeks). After CHOP, 57% achieved a complete response (CR), 37% a partial response (PR), and 6% were nonresponders (NR). At this stage, patients proving PCR-negative (n = 41) or failing to achieve a clinical response (n = 8) were excluded from rituximab treatment. Seventy-seven patients received rituximab and entered a scheduled MRD follow-up program. At the first molecular follow-up (+12 weeks), 59% had converted to PCR negativity in the BM and PB, with a further increase documented at the second control (+28 weeks) with 74% PCR negative. At the last molecular follow-up (+44 weeks), 63% of the patients remained PCR negative. At 3 years, the estimated overall survival of all patients is 95% (95% confidence interval [CI], 86-98). For patients achieving PCR-negative status following CHOP and therefore excluded from rituximab treatment, freedom from recurrence (FFR) was 52% (95% CI, 28-71). For patients treated with rituximab, a durable PCR-negative status was associated with a better clinical outcome since FFR was 57% (95% CI, 23-81) compared with 20% (95% CI, 4-46) in patients who never achieved or lost the molecular negativity (P <.001).

A distinct pathologic entity (ALK+ lymphoma) that is characterized by expression of the anaplastic lymphoma kinase (ALK) protein has recently emerged within the heterogeneous group of CD30(+) anaplastic large-cell lymphomas. Information on clinical findings and treatment outcome of ALK+ lymphoma is still limited, and no data are available concerning the value of the International Prognostic Index when applied to this homogeneous disease entity. To clarify these issues, a recently developed monoclonal antibody ALKc (directed against the cytoplasmic portion of ALK) was used to detect expression of the ALK protein in paraffin-embedded biopsies from 96 primary, systemic T/null anaplastic large-cell lymphomas, and the ALK staining pattern was correlated with morphological features, clinical findings, risk factors (as defined by the International Prognostic Index), and outcome in 78 patients (53 ALK+ and 25 ALK-). Strong cytoplasmic and/or nuclear ALK positivity was detected in 58 of 96 ALCL cases (60.4%), and it was associated with a morphological spectrum (common type, 82.7%; giant cell, 3.5%; lymphohistiocytic, 8. 6%; and small cell, 5.2%) that reflected the ratio of large anaplastic elements (usually showing cytoplasmic and nuclear ALK positivity) to small neoplastic cells (usually characterized by nucleus-restricted ALK expression). Clinically, ALK+ lymphoma mostly occurred in children and young adults (mean age, 22.01 +/- 10.87 years) with a male predominance (male/female [M/F] ratio, 3.0) that was particularly striking in the second-third decades of life (M/F ratio, 6.5) and usually presented as an aggressive, stage III-IV disease, frequently associated with systemic symptoms (75%) and extranodal involvement (60%), especially skin (21%), bone (17%), and soft tissues (17%). As compared with ALK+ lymphoma, ALK- cases occurred in older individuals (mean age, 43.33 +/- 16.15 years) and showed a lower M/F ratio (0.9) as well as lower incidence of stage III-IV disease and extranodal involvement at presentation. Overall survival of ALK+ lymphoma was far better than that of ALK- anaplastic large-cell lymphoma (71% +/- 6% v 15% +/- 11%, respectively). However, within the good prognostic category of ALK+ lymphoma, survival was 94% +/- 5% for the low/low intermediate risk group (age-adjusted International Prognostic Index, 0 to 1) and 41% +/- 12% for the high/high intermediate risk group (age-adjusted International Prognostic Index, >/=2). Multivariate analysis identified ALK expression and the International Prognostic Index as independent variables that were able to predict survival among T/null primary, systemic anaplastic large-cell lymphoma. Thus, we suggest that such parameters should be taken into consideration for the design of future clinical trials.

Purpose: Fludarabine phosphate is effective as a single agent in low-grade non-Hodgkin's lymphoma (NHL). Combined with other antineoplastic agents it enhances the antitumor effect. Our aim was to define the therapeutic efficacy and toxicity of a combination of fludarabine, cyclophosphamide and dexamethasone (FluCyD) in patients with advanced low-grade lymphoma., Patients and Methods: Twenty-five adults with pretreated advanced-stage low-grade NHL were treated with three-day courses of fludarabine 25 mg/m2/day, cyclophosphamide 350 mg/m2/day, and dexamethasone 20 mg/day, every four weeks for a maximum of six courses., Results: Of the 25 patients, 18 (72%) responded, 8 (32%) achieving CR and 10 (40%) PR. Seven were failures. The median follow-up was 21 months (5-26). Eight CR patients remain in CR after 5-21 months. Of 10 PR patients, 3 are in continuous PR without further treatment after 12, 17 and 18 months. Myelosuppression was the most prevalent toxic effect. Although severe granulocytopenia (granulocyte count nadir < 500/microliter) and thrombocytopenia (platelet count nadir < 50,000/microliter) occurred in only 10% and 16% of courses, respectively, slow granulocyte or platelet count recovery caused delay of 40% of the courses. Nine patients (36%) required discontinuation of therapy because of persistent granulocytopenia and/or thrombocytopenia: three after one course, three after 2-4 courses, and three after five courses. Thirteen infectious episodes in 11 patients complicated 11% of courses. Two of 10 patients monitored for the circulating EBV load showed increased viral load. One of these developed aggressive lymphoma. CD4+ lymphocytes declined from a pre-therapy median value of 425/microliter to 141/microliter post-treatment (P = 0.001). Non-hematologic toxicities were rare and mild., Conclusions: The combination of fludarabine with cyclophosphamide and dexamethasone is effective in pretreated advanced-stage low-grade NHL. It may broaden the range of therapeutic options in the salvage treatment of these patients. The main toxicity of this combination is prolonged myelosuppression that may cause treatment delay or withdrawal. The benefit of adding granulocyte colony-stimulating factor, particularly in patients with poor marrow reserve, needs to be investigated.

Castleman's disease is a rare B cell lymphoproliferative disorder related to excess interleukin-6 (IL-6)-like activity. Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8), which encodes a functional cytokine (vIL-6), has been found in some patients with Castleman's disease. Lymph nodes from 14 HIV-seronegative Castleman's disease patients were compared to hyperplastic lymph nodes from 25 HIV-seronegative patients as well as Kaposi's sarcoma lesions from 48 patients for KSHV infection and vIL-6, human IL-6, and Epstein-Barr virus EBER expression. While all Kaposi's sarcoma tissues examined were polymerase chain reaction-positive and all control lymph nodes were polymerase chain reaction-negative for KSHV, none had detectable vIL-6 expression. Six of 14 (43%) Castleman's tissues were positive for KSHV by polymerase chain reaction and all 6 had evidence of vIL-6 expression by immunohistochemistry. vIL-6-positive Castleman's disease patients generally had the multicentric plasma cell variant form of the disease and had a rapidly fatal clinical course frequently associated with autoimmune hemolytic anemia and gammopathy. In contrast, 7 (88%) of the 8 vIL-6-negative Castleman's disease patients had localized disease and have remained disease-free after therapy. KSHV vIL-6 expression appears to be limited to hematopoietic cells and is not present in Kaposi's sarcoma spindle cells. These data suggest that Castleman's disease is a syndrome of multiple etiologies involving aberrant IL-6 activity from either endogenous or viral sources.

From March 1993 to October 1993, 20 consecutive, newly diagnosed acute promyelocytic leukemia (APL) patients from 13 Italian institutions entered in a pilot study named AIDA, combining all-trans retinoic acid (ATRA) with idarubicin (IDA). ATRA was administered orally beginning on the first day of induction at the dosage of 45 mg/m2/d until complete remission (CR), whereas IDA was administered intravenously at the dosage of 12 mg/m2/d on days 2, 4, 6, and 8 of the induction. Patients who achieved CR were consolidated with 3 courses of chemotherapy without ATRA; thereafter, they were followed up for molecular and hematologic CR. The median age was 35.3 years (range, 6.5 to 67.6 years); 8 patients were males and 12 females; 4 had the hypogranular variant of APL (M3v), and 4 (2 with M3v) presented with leukocyte counts > or = 10,000/microL. Molecular analysis for the promyelocytic leukemia-retinoic acid receptor alpha (PML-RAR alpha) hybrid gene at diagnosis was performed in 16 patients by means of reverse transcription-polymerase chain reaction (RT-PCR) analysis, and all were RT-PCR+ for the hybrid gene. In the remaining 4 patients, the cytogenetic study showed the presence of the t(15;17). After a median time of 36 days (range, 28 to 52 days) 18 (90%) patients achieved CR; the remaining 2 patients died 12 and 34 days after diagnosis from myocardial infarction caused by fungal myocarditis and from massive hemoptysis, respectively. ATRA syndrome was observed in only 2 patients, and, after the prompt discontinuation of ATRA and initiation of dexamethasone, both recovered from the syndrome. However, after recovering, 1 patient achieved CR, whereas the other died at day 34 because of massive hemoptysis; other side effects were very limited. At recovery from the third consolidation course, only 3 of 14 (21.4%) tested patients were RT-PCR+ for the PML-RAR alpha hybrid gene. Of these, 2 relapsed shortly afterwards; however, in the last patient, the PML-RAR alpha disappeared at successive testing performed 2 months later. As of September 30, 1995, after a median follow-up period from diagnosis of 27 months (range, 24 to 31 months), the overall survival and event-free survival durations are 85% and 69%, respectively; moreover, 14 of 18 (78%) patients who achieved CR are still alive and in first molecular and hematologic CR. Of the 4 relapsed patients, 3 achieved a second CR with ATRA and, after further treatment, are now in molecular and hematologic CR after 4+, 16+, and 17+ months from the second CR. These results indicate that (1) the AIDA protocol is highly effective in treating APL; (2) after 3 consolidation courses, the majority of patients who achieved CR are RT-PCR- for the hybrid gene PML-RAR alpha; (3) the persistence of an RT-PCR positivity for the PML-RAR alpha hybrid gene after 3 consolidation courses is indicative of early relapse, thus these patients still require additional treatment. These results have prompted the Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (GIMEMA) to initiate, in cooperation with the Associazione Italiana di Ematologia ed Oncologia Pediatrica and some European Organization for Research and Treatment of Cancer (EORTC) centers, a new multicentric clinical trial named AIDA LAP 0493 for the treatment of adult and pediatric APL patients. All patients are considered eligible if APL diagnosis is confirmed with molecular or cytogenetic studies for PML-RAR alpha hybrid gene or t(15;17) and are enrolled to receive the same induction and consolidation therapy of this pilot study. After consolidation, patients who are RT-PCR- for PML-RAR alpha hybrid gene are randomized to four arms, whereas patients who are RT-PCR+ after consolidation undergo, if eligible, an allogenic transplantation procedure.

Background: A prospective study was conducted to assess the efficacy and toxicity of a salvage regimen consisting of CCNU, Melphalan, and VP-16 (CAV) given at 28-day intervals in patients with Hodgkin's disease (HD) relapsing after primary therapy or refractory to the alternating MOPP/ABVD regimen., Patients and Methods: This study included 58 patients (median age: 34 years), with resistant or relapsing HD. Primary therapy had consisted of alternating MOPP/ABVD (81%) or MOPP alone (19%); 38% of patients were relapsing from prior complete remission (CR) while 62% had resistant disease. Extranodal disease was present in 55% and B-symptoms in 72% of patients; one-fifth had bulky disease and/or bone marrow involvement. The CAV was used as first salvage in half of the patients., Results: Complete remission was obtained in 17 patients (29%); unfavorable factors for CR in univariate analysis were the presence of bulky disease and the failure to achieve CR with prior therapy. Nine patients (53% of remitters) have subsequently relapsed with a 10-month median duration of CR. The 3-year overall survival after CAV was 25% with an 18-month median survival; significant differences in survival were found according to the extent of disease, the presence of B-symptoms and the HD status (prior sensitive or resistant disease, first or subsequent relapse). Seven patients are long-term remitters (12%), and one of them has been given high-dose chemotherapy and autologous bone marrow transplantation at relapse after CAV. The CAV toxicity was mostly hematological; severe pancytopenia occurred in six cases with two cases of fatal infections and one of fatal hemorrhage., Conclusion: CAV therapy was moderately effective as third-line salvage in patients with HD resistant to alternating MOPP/ABVD or previously given two different regimens for relapse; the toxicity was mostly hematological and supportive therapy was needed in one-third of the patients.

Background: A prospective study was conducted to assess (a) the long-term results and toxicity of the alternating MOPP/ABVD regimen in advanced Hodgkin's disease; (b) the prognostic value of pretreatment variables and of drug dose intensity., Patients and Methods: A total 138 consecutive patients with advanced Hodgkin's disease entered this study; patient selection included stages IIB (33% of total), IIIB (26%), IV (25%), and stages IIA-IIIA (16%) with bulky disease and pulmonary hilum involvement. The MOPP/ABVD program was delivered in an 8-month program; adjuvant radiotherapy on sites of bulky disease was delivered in 24 patients., Results: Complete remission was obtained in 106 (77%) patients; significant factors for CR in univariate analysis were stage, symptoms, histology, and bone marrow involvement. The five-year relapse-free survival (RFS) was 83%; in a multivariate analysis, histology only correlated with RFS (p = 0.04). The five-year freedom from tumor mortality and overall survival (OS) were 79% and 67%, respectively. An adverse prognostic significance for OS was observed for B symptoms and bone marrow involvement. The median percentage of relative dose intensity (RDI) was as follows: Adriamycin 86, mechlorethamine 85, vincristine 73, vinblastine 84, bleomycin 79, procarbazine 74, dacarbazine 81. No significant association was found between RDI and clinical outcome. No severe pancytopenia or life-threatening complications occurred during therapy., Conclusions: Alternating MOPP and ABVD cured more than 65% of patients with advanced HD; acute and late toxicity were acceptable. Prognostic analysis defined subgroups with a lower chance of cure which may deserve a more intensive initial therapy.

Background: Controversy still exists over the optimal management of early-stage Hodgkin's disease (HD); presentation features may have a different prognostic impact according to initial therapy, and long-term toxicity must be fully evaluated., Patients and Methods: This study included 164 patients with stage IA-IIA HD treated with radiotherapy (RT) alone or combined radio- and chemotherapy (CT) according to presenting features and their attendant prognostic significance. The RT group included 88 patients with favorable prognostic features; the combined modality group included 76 patients with one or more unfavorable features. In the RT group, 85% of patients received extended-mantle or STNI; in the combined modality group, RT consisted of mantle- (49%), extended mantle- (37%), and involved-field irradiation (14%); CT consisted of 6 cycles of MOPP before 1984; 3 cycles of ABVD were substituted for MOPP thereafter., Results: Complete remission was obtained in 94% and 99% of patients of the RT and combined modality groups, respectively. The 10-year actuarial relapse-free survival (RFS) in the RT group was 62% and was influenced by stage (p = 0.04) and histology (p = 0.01); in the combined modality group, RFS was 88% and was influenced by the presence of bulky disease. Overall survival and tumor mortality between the therapy groups were comparable. RT-related toxicity consisted of mediastinal fibrosis (8 cases), myelitis (3), hypothyroidism (2); other long-term events included 2 cases of acute leukemia in the combined MOPP and RT group. Altogether, 8 of 20 patients who died were in their first complete remission., Conclusions: In stage IA-IIA HD, the combined modality therapy reduced the risk of relapse compared to radiation alone; long-term toxicity of RT was not negligible and relapses could occur late.

This paper analyzes the clinicopathological features of 31 consecutive adult patients with lymphoblastic lymphoma (LBL) and reports on our experience in treating them with two successive multidrug programs analogous to those used in acute lymphoblastic leukemia (ALL) in adults. Protocol 1 (18 patients) consisted of an intensive four-drug induction therapy (daunorubicin, cyclophosphamide, vincristine, prednisone), CNS prophylaxis (cranial irradiation and intrathecal methotrexate), and continuous maintenance with methotrexate and mercaptopurine for three years, with periodical reinductions with vincristine and prednisone. Protocol 2 (13 patients) included a similar induction regimen (doxorubicin instead of daunorubicin, dexamethasone instead of prednisone), followed by post-remission intensification with alternating courses of non-cross-resistant agents (amsacrine and high dose cytarabine; vincristine, cyclophosphamide and doxorubicin; etoposide and conventional dose cytarabine) given in a cyclical eight-month program. CNS prophylaxis consisted of intrathecal methotrexate and systemic high-dose cytarabine. The patient characteristics of the two therapy groups were comparable. The complete remission (CR) rate for both groups was 77%, with a median overall and relapse-free survival of 18 and 29 months, respectively. The three-year overall survival of complete remitters was 59%. No correlation was found between CR rate and age, mediastinal, or bone marrow involvement. Stage I disease had a significantly higher CR rate (100%) than did stages II-IV (64%). Leukemic evolution occurred in 32% of cases, within a median time of 11 months from diagnosis; meningeal disease developed in six cases (19%); in four of them leukemia was concomitant. No significant differences were found between the two successive treatments for CR rate, survival, CNS relapses or toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

We have analyzed the distribution and prognostic significance of terminal deoxynucleotidyl transferase (TdT) and adenosine deaminase (ADA) in connection with conventional cytology, cytogenetics, response to therapy, and survival. The study population consisted of 78 patients with AML, 44 patients with Ph1 + CML in chronic phase, and 35 adult patients with Ph1 + CML in blastic phase, among which 5 cases presented as Ph1 + acute leukemia. Nine percent of the AML cases were positive for TdT and were characterized by a high percentage of blast cells in bone marrow, myeloid features by cytochemistry and absence of the Philadelphia chromosome. The median ADA values of the TdT+ AML cases were several times higher than those obtained for the TdT- cases. The survival calculated for the two groups of AML cases subdivided according to ADA levels was significantly longer (p less than 0.025) for the patients with low levels of ADA (less than 250 U/10(8) cells). In chronic phase of CML, TdT was absent and ADA values were increased over normal controls only in cases with early signs of transformation. In blastic phase, 31% of the 35 cases were positive for TdT, and ADA values were significantly higher (p less than 0.001) in TdT+ than TdT- cases. The survival calculated from the onset of transformation was significantly longer for the TdT+ acute phase (10.4 mo) compared to the TdT- patients (4.8 mo; p less than 0.025). Four cases presenting as Ph1 + acute leukemia were TdT+ and had elevated levels of ADA; 3 of them responded to ALL therapy, reverting to a stable phase of CML.

The authors treated a total of 82 patients with Ph'-positive chronic myelogenous leukemia (CML) with recombinant interferon alpha-2b (IFN alpha-2b). Sixty-five patients in chronic phase (CP), 28 of whom were untreated and 37 pretreated, and nine patients in accelerated phase (AP), were started on IFN three times a week. Patients in CP were randomized to receive 2 or 5 X 10(6) IU/m2, while patients in AP were all given the dose of 5 X 10(6) IU/m2, in addition to concomitant chemotherapy. Patients in CP who were unresponsive to the lower dose were crossed to the higher dose. Of 63 evaluable patients in CP, 43 (68%) responded, 29 (46%) achieved complete hematologic response (CHR), and 14 (22%) achieved partial hematologic response (PHR). The response rate appeared to be significantly influenced by the IFN dose in pretreated patients. Of the nine patients in AP, two attained PHR and one CHR. More recently, eight previously untreated CP cases were submitted to daily IFN administration at doses from 2 to 5 X 10(6) IU/m2. This daily schedule was also applied to patients who had obtained, with the intermittent treatment, a PHR persisting unmodified for six months (nine patients) or an unstable CHR (five patients). Seven of the eight previously untreated patients, and five of the nine PHR patients crossed to daily IFN reached CHR. In the total series of previously untreated patients, the response rate proved to be significantly influenced by the initial risk status. Cytogenetic improvement was seen in 37 of 53 responders (70%) treated for more than 3 months, the median of Ph'-positive cells declining from 100% to 65% (range 0% to 95%). Complete suppression of Ph' chromosome was observed in one case. The cytogenetic response was persistent for over 6 months in 21 patients, but the lowest value of Ph' positivity was usually unstable. At a median follow-up of 56 weeks, 23 of 36 (64%) CHR patients remain in continued disease control with IFN. A blastic transformation (BT) occurred in seven of 21 unresponsive patients and in one of the 36 CHR patients. The authors' data confirm that IFN alpha-2b, especially at daily doses, is effective in inducing clinical and cytogenetic response in a good proportion of patients with CML in the benign phase. Longer follow-ups will define the exact influence of this agent on the natural course of the disease.