Rearrangement of the c-myc oncogene with heavy-chain immunoglobulin enhancer in tumor DNA from an acute lymphoblastic leukemia patient.

The DNA obtained from the leukemia cells of an acute lymphoblastic leukemia (ALL, L3 type) with a pre-B-phenotype and a typical t(8;14) chromosomal translocation showed a rearrangement juxtaposing the c-myc gene and the immunoglobulin (Ig) heavy-chain gene enhancer. This abnormality was only present in the leukemia cells of the patient and correlated with the clinical course of the disease. The breakpoint on chromosome 8 occurred within c-myc intron 1, between 790 and 638 base pairs upstream of c-myc exon 2. This breakpoint position was the nearest to the c-myc exon 2 so far described in Burkitt's type lymphoma-leukemias, and it mapped very near to the location of a major cryptic promoter used by truncated c-myc genes. In spite of what was detected in a human lymphoma cell line (Manca) carrying a similar rearrangement, in this case the amount of c-myc transcript was not increased compared to an Epstein-Barr virus-transformed normal lymphoblastoid cell line obtained from the same patient. This may in part be due to the breakpoint position and to the fact that the efficiency of the major cryptic promoter present within the first intron could have been affected by the translocation event. Finally, as previously suggested by others, the phenotype expressed by the leukemia cells supported the notion that this particular type of rearrangement (linking together the c-myc gene and the Ig heavy-chain gene enhancer element) may be associated with a subgroup of B-ALLs showing an immunologic phenotype relatively more immature than that of classical B-ALL.

Capecitabine with/without mitomycin C: results of a randomized phase II trial of second-line therapy in advanced biliary tract adenocarcinoma.

PURPOSE: Advanced biliary tract adenocarcinoma (BTA) is a rare tumor with a poor prognosis. Since no standard salvage chemotherapy regimen exists, we explored the activity of capecitabine alone or combined with mitomycin C. METHODS: Patients aged 18-75 years and with KPS >50, with pathological diagnosis of BTA stratified based on site and stage of disease, were randomized to receive capecitabine 2000 mg/m(2) day 1-14 alone (ARM A) or in combination with mitomycin C 6 mg/m(2) day 1 (ARM B) as second-line therapy. Cycles were repeated in both arms every 3 weeks. Tumor assessment was performed every 2 months. The primary endpoint was the probability of being progression free at 6 months (PFS-6) from treatment start. According to the Fleming design, the study aimed to enroll 26 pts per arm. An exploratory endpoint was to assess thymidylate synthase (TS) and thymidine phosphorylase (TP) expression, as biomarkers predictive for clinical outcomes of capecitabine treatment. RESULTS: Between October 2011 and 2013, 57 metastatic pts were enrolled: ARM A/B 28/29. Accordingly, 55 (26/29) pts were assessable for the primary endpoint: 2 (8%) ARM A and 3 (10%) ARM B pts were PFS-6. Main G3-4 toxicities were: hand-foot syndrome and transaminitis in 4/0%, and thrombocytopenia, diarrhea and fatigue in 0/3% of pts. No statistically significant correlation was found between TS or TP expression and pts' outcome. CONCLUSIONS: Since capecitabine yielded a disappointing outcome and the addition of mitomycin C did not improve the results, new therapeutic strategies need to be explored to improve survival in this disease setting.

High-dose sequential (HDS) chemotherapy with blood and marrow cell autograft as salvage treatment in very poor prognosis, relapsed non-Hodgkin's lymphoma.

We tested the feasibility and efficacy of a novel high-dose sequential chemoradiotherapy programme (HDS) in 14 relapsed or refractory non-Hodgkin's lymphoma patients with very poor prognostic features, i.e. transformed histology, marrow invasion, low performance status. This regimen included the sequential administration of high-dose cyclophosphamide (CY) 7 g/m2 followed by high-dose methotrexate (MTX) 8 g/m2 and high-dose VP16 2 g/m2 and finally by total body irradiation (TBI)-melphalan and autograft of bone marrow and peripheral blood progenitor cells. No hemopoietic growth factor support was employed in any phase. There was one treatment-related death during the high-dose phase; three other patients did not complete the programme. All 10 patients concluding the programme achieved complete remission, with four patients in complete clinical remission at a median follow up of 34 months. Median overall survival was 27 months and median failure-free survival (FFS) was 12 months. Twenty-six well comparable patients received conventional salvage therapy during the same period. Their projected median overall survival (8 months) and median FFS (4 months) were shorter than in the HDS group (p = 0.06 for overall survival and p = 0.03 for FFS). Thus, HDS is a feasible programme and may offer superior results than conventional therapy in poor-prognosis NHL patients.

G-CSF administration following peripheral blood progenitor cell (PBPC) autograft in lymphoid malignancies: evidence for clinical benefits and reduction of treatment costs.

Clinical value and costs of G-CSF administration following autograft with mobilized peripheral blood progenitor cells (PBPC) were evaluated in two sequential groups of 20 patients each, treated for lymphoid neoplasms in the period February 1993 to January 1996. One group was given G-CSF (Filgrastim) (5 microg/kg/day), starting on day +1 until ANC was > 500/microl, the other received no G-CSF. All patients were conditioned with mitoxantrone 60 mg/m2 + L-PAM 180 mg/m2 and received large numbers of PBPC (median of 12 and 13 x 10(6) CD34+/kg, respectively). The median time to ANC > 500/microl was 10 days in the G-CSF group vs 14 days in controls (P < 0.0001). G-CSF was associated with a slightly faster platelet recovery (11 vs 13 days to plts > 20000/microl, P = 0.09). Median duration of fever (2.5 vs 5 days, P = 0.028), nonprophylactic antibiotics (8 vs 11 days, P = 0.019), and post-transplant hospitalization (13 vs 16 days, P = 0.0028) were also significantly reduced. The average cost per treatment in the G-CSF group amounted to about US$18241 as compared to US$21868 in the control group, implying a cost reduction of approximately 16%. Thus, G-CSF reduced morbidity with cost containment, supporting its use even if autograft is performed with large quantities of PBPC.

N-ras mutations in myeloid leukemias.

The presence of mutations activating the N-ras gene was investigated by the polymerase chain reaction technique in twenty patients with acute myeloblastic leukemia (AML) at onset and in four patients with Ph1 positive chronic myelogeneous leukemia (CML) either in chronic phase or in blast crisis. Four remission samples and four relapses from the AML cases were also studied. Mutations were found in five out of twenty (25%) untreated AML cases at onset. No mutations were detected in the complete remission samples, two of them with N-ras mutations during the leukemic phase. Two out of the four leukemia relapses were positive for the same N-ras mutation shown at presentation, whereas no new mutations were found in the other two initially negative cases. An N-ras mutation appeared during the blast crisis of one of the four CML, which were all negative during the chronic phase. In conclusion, whereas some data appear to be consistent with a role of the N-ras mutations as initiating events in myeloid leukemias, in other cases N-ras activation seems to represent a factor involved in progression. These data suggest that a partial overlapping between initiation and progression factors could exist in naturally occurring tumors.

Immune complexes in aplastic anaemia.

Immune complexes (IC) have been detected in nine out of 15 patients presenting with idiopathic aplastic anaemia using the polymorphonuclear neutrophil immunohistochemical technique. Immunosuppressive treatment undertaken in one patient produced a gradual recovery, the bone marrow repopulation being paralleled by the disappearance of IC. The significance of IC in aplastic anaemia and the relationship with possible pathogenesis and therapy of the disease are discussed.

Treatment of acute myeloid leukaemia patients with recombinant interleukin 2: a pilot study.

Twelve patients with acute myeloid leukaemia (AML) with evidence of resistant disease were treated with recombinant interleukin 2 (rIL2) given intravenously by continuous infusion. No objective response to rIL2 alone was documented in the seven patients with advanced disease (20-90% resistant blasts in the marrow), except for a partial response to rIL2 plus chemotherapy in one. Of the five patients with limited disease (8-15% marrow blasts), three obtained a complete disappearance of the blasts following two to four 5d courses of rIL2 alone. One patient persists in fourth complete remission (CR) 30 months later, another obtained a third CR for 4 months, and the last remained in third CR for 9 months before relapsing. This latter patient achieved a fourth CR with low-dose cytarabine. The remissions have been maintained with low-dose monthly courses of rIL2 given on an out-patient basis. Two AML did not respond to rIL2 alone; one, however, obtained a fourth CR with chemotherapy and rIL2. Administration of rIL2 was accompanied by organomegaly and leucocytosis, with a frequent lymphocytosis and increase in eosinophils and large granular lymphocytes, both in the blood and in the marrow. Side effects, though often severe, were controllable using a daily dose escalating protocol and never required intensive care treatment. The results of this pilot study indicate that treatment of AML patients with rIL2 is feasible and may result in the disappearance of chemotherapy-resistant blasts in patients with limited but detectable disease. Further controlled trials in AML in CR appear warranted.

Idiopathic myelofibrosis: a possible role for immune-complexes in the pathogenesis of bone marrow fibrosis.

Sixteen patients with idiopathic myelofibrosis (IM) have been investigated with respect to the possibility that immune mechanisms may be of importance in the pathogenesis of bone marrow fibrosis. The following points appear relevant: (1) immune-complexes (IC) are detectable with different techniques in a high percentage of patients with IM. Their presence is associated with evidence of bone-marrow histological markers of immune activity. (2) IgG is the main Ig class in the composition of IM IC. The results obtained favour the hypothesis that autoimmune mechanisms are involved in IM patients.

Thrombocytopenia in acute leukaemia patients treated with IL2: cytolytic effect of LAK cells on megakaryocytic progenitors.

In vivo administration of recombinant interleukin 2 (IL2) has been associated, in acute leukaemia as well as in other tumours, with a variable degree of thrombocytopenia. In two patients with acute myeloid leukaemia who showed a progressive and severe fall in platelet count during daily continuous i.v. infusion of IL2, we assessed whether peripheral blood IL2-generated lymphokine activated killer (LAK) lymphocytes could affect growth of the autologous bone marrow megakaryocytic progenitor cell compartment (CFU-MK) in vitro. Following overnight pre-incubation in liquid culture of the marrow cells with autologous LAK effectors, there was an almost complete abrogation of the CFU-MK colony growth (97% and 89% inhibition). Pre-incubation in the presence of a monoclonal antibody to tumour necrosis factor alpha (TNF) completely reversed the inhibitory effect. The role played by TNF was confirmed by the finding that recombinant TNF caused a dose-dependent inhibition of the growth of CFU-MK. IL2 alone was ineffective. These results suggest that the often severe thrombocytopenia observed in patients with acute leukaemia treated with IL2 is at least partly due to autologous LAK cells activated in vivo following the administration of IL2.

High prevalence of autoimmune phenomena in hepatitis C virus antibody positive patients with lymphoproliferative and connective tissue disorders.

The aim of this study was to investigate whether hepatitis C virus (HCV) may perturb the immune system towards autoreactivity. We studied the relationship between the prevalence of anti-HCV and the presence of laboratory and/or clinical autoimmune features in 300 patients: lymphoid malignancies (167) and autoimmune disorders (connective tissue diseases 100; idiopathic thrombocytopenic purpura (ITP) 33). As a control, hepatitis B surface antigen (HBV) and anti-hepatitis B core antigen (anti-HBc) were related to the same parameters. Anti-HCV and anti-HBV were detected in 68/300 (22.6%) and 70/300 (24.6%) patients, respectively. HCV prevalence was 18% in lymphoproliferative disorders (anti-HBc 28.1%) and 26% in connective tissue disease (anti-HBc 16.3%). Among ITP cases, 12/33 (36.4%) were anti-HCV+ and 10/33 (30.3%) anti-HBc+. In 24/30 (80%) anti-HCV+ patients with lymphoproliferative disorders at least one serologic or clinical autoimmune abnormality was detected. To the contrary, only 10/45 (22.2%) anti-HBc+ patients with lymphoproliferative disorders had at least one serologic or clinical abnormality (P < 0.0001). A statistically significant correlation was observed between HCV prevalence and the number of autoimmune alterations in both lymphoproliferative and connective tissue disorders, which was not found for anti-HBc. These data suggest that HCV may skew the immune system toward the production of autoantibodies and also support the possibility that some cases of ITP may be linked to both HCV and HBV infection.

Immune-complexes (IC) in idiopathic neutropenia.

The presence of immune-complexes (IC) and antipolymorphonuclear neutrophil (PMN) autoantibodies was investigated in 28 patients with chronic idiopathic neutropenia and normal or hypercellular bone marrow, 19 with a metamyelocyte arrest and 9 with more dysplastic features. The in vivo interaction between IC and PMN membrane receptors was evaluated by means of the PMN immunohistological technique. Circulating IC was evaluated with the C1q and rheumatoid factor agglutination inhibition techniques. An anti-PM autoantibody activity was investigated by challenging Fab obtained from the sera of 22 patients with PMN from normal donors. IC were detected in a high percentage of patients; in no case could an anti-PM autoantibody activity be seen. Most patients with a metamyelocyte arrest, but only 1 with more dysplastic features, were IC+. During a follow-up period of l2-52 months, none of the patients with a metamyelocyte arrest (IC+) developed anaemia, thrombocytopenia or leukaemia, while anaemia and thrombocytopenia were almost the rule in the clinical course of dysplastic bone marrow IC- patients: 2 of them developed acute myeloblastic leukaemia.

Differences in the in vitro growth pattern of fresh and cryopreserved granulo-monopoietic precursors.

A study of the in vitro growth model of human granulo-monopoietic precursors (CFU-GM) before and after cryopreservation using both leukocyte feeder layers and GCT conditioned medium as the source of colony stimulating activity (CSA) is reported. The number of colonies produced with fresh cells was linearly related to the amount of marrow seeded with both CSA sources, whereas after cryopreservation this was true with feeder layers, and with GCT only at relatively high cell concentrations. This might indicate the production of granulopoietic stimulators on the part of a second population that is at least partly resistant to freezing. It seems more likely, however, that these results depend mainly on a sublethal damage to CFU-GM induced by freezing, thus making the cells hyporesponsive to some forms of CSA, such as those contained in GCT conditioned medium.

Phase II study of cisplatin, gemcitabine and 5-fluorouracil in advanced pancreatic cancer.

BACKGROUND: The aim of this study was to determine the activity of the combination of cisplatin, gemcitabine and 5-fluorouracil (5-FU) as therapy for metastatic or locally advanced inoperable pancreatic adenocarcinoma. PATIENTS AND METHODS: Patients with histologically proven advanced or metastatic pancreatic adenocarcinoma received first-line chemotherapy comprising cisplatin (20 mg/m2 on days 1, 8, 15, 22, 29 and 36), gemcitabine (1000 mg/m2 on days 1, 8, 29 and 36) and 5-FU (200 mg/m2 as continuous infusion on days 1-42) every 56 days. RESULTS: A total of 34 patients were studied. Eighty courses were administered (median two courses per patient). Among 32 patients evaluable for response, two patients had a complete response and four a partial response for an overall response rate of 19% (95% confidence interval 7% to 36%). Thirteen patients had stable disease (40%) and 13 progressed. Median progression-free survival was 4.7 months, median survival 9.0 months and 26% of patients achieved 1-year survival. Ten of 25 patients (40%) with pain at presentation had a sustained reduction of analgesic consumption. The principal grade 3/4 toxicities were neutropenia, thrombocytopenia, anaemia and mucositis, occurring in 24%, 21%, 9% and 3% of patients. CONCLUSION: This schedule seems well tolerated and active in pancreatic cancer and worthwhile of further evaluation.

The nature of the B lymphocyte in B-chronic lymphocytic leukemia.

We have analyzed phenotypic, functional, and molecular properties of B-chronic lymphocytic leukemia (B-CLL) cells as compared to normal B cell differentiation stages and/or subsets. The possibility that the target B cell population transformed by the I primary oncogenic event(s) belongs to the normal CD5+ B cell subset from B mantle zone of secondary follicles is highly likely on phenotypic grounds. Though the genes responsible for the primary oncogenic event are presently unknown, a number of functional and molecular findings indicate that the end-product of their transforming activity is a cell frozen in the G0 phase of the cell cycle. This cell has several abnormalities that prevent an appropriate mitogenic response and presents a pattern of apoptosis-related gene expression that hinders apoptotic death. Pivotal to this apoptosis-escaping capacity is the expression of Bcl-2. We suggest that the increased expression of Bcl-2 together with an asynchronism between the expression of Bcl-2, c-myc, and APO1/Fas gene products shift the cellular balance away from apoptosis thereby helping the progressive accumulation in G0 of malignant CD5+ B cells.

Probability of cure in elderly Hodgkin's disease patients.

BACKGROUND: Elderly Hodgkin's disease patients have a poor prognosis. The question arises whether these patients need aggressive treatment or a palliative strategy. So far, as a consequence of the scarcity of trials designed for them, useful information can be obtained only by retrospective analyses. METHODS: We retrospectively studied clinical data from 567 patients recorded from 1982 to 1989 in the Piemonte Hodgkin's Disease Register (PHDR). The 65 patients over 65 years of age were compared to younger ones. We analyzed the role of disease independently of confounding variables, mainly inadequacy of staging and/or treatment, comorbidity and toxicity. RESULTS: In the elderly comorbidity was as high as 35%. Forty elderly patients (60%) entered a suboptimal plan with a low degree of aggressivity, which was different from the usual PHDR protocol. Elderly patients also had a high proportion of subsequent protocol interruptions (25%). Chemotherapy dose intensity was negatively affected by advanced age (p < 0.01 after both 3 and 6 courses of chemotherapy). Toxic deaths were significantly higher in elderly patients than in younger ones (14% vs 1%; p < 0.05). CR rates, overall survival (OS), disease-specific survival (DSS) and event free survival (EFS) were all significantly influenced by age (p < 0.01). Relapse-free survival (RFS) in patients achieving CR did not differ according to age class (77% vs 60%; p = ns). RFS was better in elderly patients entering the PHDR protocols than in those following an alternative plan (75% vs 54%; p = 0.04); however, elderly patients treated according to PHDR guidelines showed a higher incidence of toxic deaths than those treated less aggressively (23% vs 8%). The two groups had similar EFS (36% vs 24%; p = ns). CONCLUSIONS: Elderly patients who achieve CR can have good RFS and cure is possible, but the toxic cost of conventional strategies is unacceptable and selected strategies still must be found.