Ruxolitinib Adherence in Myelofibrosis and Polycythemia Vera: the "RAMP" Italian multicenter prospective study.

Ruxolitinib is beneficial in patients with myelofibrosis (MF) and polycythemia vera (PV). Information on ruxolitinib adherence is scant. The Ruxolitinib Adherence in Myelofibrosis and Polycythemia Vera (RAMP) prospective multicenter study (NCT06078319) included 189 ruxolitinib-treated patients. Patients completed the Adherence to Refills and Medications Scale (ARMS) and Distress Thermometer and Problem List (DTPL) at the earliest convenience, after registration in the study, and at later timepoints. At week-0, low adherence (ARMS > 14) and high distress (DT ≥ 4) were declared by 49.7% and 40.2% of patients, respectively. The main reason for low adherence was difficult ruxolitinib supply (49%), intentional (4.3%) and unintentional (46.7%) non-take. In multivariable regression analysis, low adherence was associated to male sex (p = 0.001), high distress (p < 0.001), and treatment duration ≥ 1 year (p = 0.03). Over time, rates of low adherence and high distress remained stable, but unintentional non-take decreased from 47.9% to 26.0% at week-48. MF patients with stable high adherence/low distress were more likely to obtain/maintain the spleen response at week-24. Low adherence to ruxolitinib represents an unmet clinical need that require a multifaceted approach, based on reason behind it (patients characteristics and treatment duration). Its recognition may help distinguishing patients who are truly refractory and those in need of therapy optimization.

MacroH2A1.1 as a crossroad between epigenetics, inflammation and metabolism of mesenchymal stromal cells in myelodysplastic syndromes.

Ineffective hematopoiesis is a hallmark of myelodysplastic syndromes (MDS). Hematopoietic alterations in MDS patients strictly correlate with microenvironment dysfunctions, eventually affecting also the mesenchymal stromal cell (MSC) compartment. Stromal cells are indeed epigenetically reprogrammed to cooperate with leukemic cells and propagate the disease as "tumor unit"; therefore, changes in MSC epigenetic profile might contribute to the hematopoietic perturbations typical of MDS. Here, we unveil that the histone variant macroH2A1 (mH2A1) regulates the crosstalk between epigenetics and inflammation in MDS-MSCs, potentially affecting their hematopoietic support ability. We show that the mH2A1 splicing isoform mH2A1.1 accumulates in MDS-MSCs, correlating with the expression of the Toll-like receptor 4 (TLR4), an important pro-tumor activator of MSC phenotype associated to a pro-inflammatory behavior. MH2A1.1-TLR4 axis was further investigated in HS-5 stromal cells after ectopic mH2A1.1 overexpression (mH2A1.1-OE). Proteomic data confirmed the activation of a pro-inflammatory signature associated to TLR4 and nuclear factor kappa B (NFkB) activation. Moreover, mH2A1.1-OE proteomic profile identified several upregulated proteins associated to DNA and histones hypermethylation, including S-adenosylhomocysteine hydrolase, a strong inhibitor of DNA methyltransferase and of the methyl donor S-adenosyl-methionine (SAM). HPLC analysis confirmed higher SAM/SAH ratio along with a metabolic reprogramming. Interestingly, an increased LDHA nuclear localization was detected both in mH2A1.1-OE cells and MDS-MSCs, probably depending on MSC inflammatory phenotype. Finally, coculturing healthy mH2A1.1-OE MSCs with CD34+ cells, we found a significant reduction in the number of CD34+ cells, which was reflected in a decreased number of colony forming units (CFU-Cs). These results suggest a key role of mH2A1.1 in driving the crosstalk between epigenetic signaling, inflammation, and cell metabolism networks in MDS-MSCs.

High-density neutrophils in MGUS and multiple myeloma are dysfunctional and immune-suppressive due to increased STAT3 downstream signaling.

To understand neutrophil impairment in the progression from MGUS through active MM, we investigated the function of mature, high-density neutrophils (HDNs), isolated from peripheral blood. In 7 MM, 3 MGUS and 3 healthy subjects by gene expression profile, we identified a total of 551 upregulated and 343 downregulated genes in MM-HDN, involved in chemokine signaling pathway and FC-gamma receptor mediated phagocytosis conveying in the activation of STAT proteins. In a series of 60 newly diagnosed MM and 30 MGUS patients, by flow-cytometry we found that HDN from MM, and to a lesser extend MGUS, had an up-regulation of the inducible FcγRI (also known as CD64) and a down-regulation of the constitutive FcγRIIIa (also known as CD16) together with a reduced phagocytic activity and oxidative burst, associated to increased immune-suppression that could be reverted by arginase inhibitors in co-culture with lymphocytes. In 43 consecutive newly-diagnosed MM patients, who received first-line treatment based on bortezomib, thalidomide and dexamethasone, high CD64 could identify at diagnosis patients with inferior median overall survival (39.5 versus 86.7 months, p = 0.04). Thus, HDNs are significantly different among healthy, MGUS and MM subjects. In both MGUS and MM neutrophils may play a role in supporting both the increased susceptibility to infection and the immunological dysfunction that leads to tumor progression.

The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Accuracy of physician assessment of treatment preferences and health status in elderly patients with higher-risk myelodysplastic syndromes.

Higher-risk myelodysplastic syndromes (MDS) are rarely curable and have a poor prognosis. We investigated the accuracy of physicians' perception of patients' health status and the patients' preferences for involvement in treatment decisions. We examined 280 newly diagnosed higher-risk elderly MDS patients paired with their physicians. Survey tools included the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) and the Control Preference Scale. Overall concordance was 49% for physician perception of patient preferences for involvement in treatment decisions. In 36.4% of comparisons there were minor differences and in 14.6% there were major differences. In 44.7% of the patients preferring a passive role, physicians perceived them as preferring an active or collaborative role. Absence of the patient's request for prognostic information (P=0.001) and judging the patient as having a poor health status (P=0.036) were factors independently associated with the physicians' attitude toward a lower degree of patient involvement in clinical decisions. Agreement on health status was found in 27.5% of cases. Physicians most frequently tended to overestimate health status of patients who reported low-level health status. The value of decision aid-tools in the challenging setting of higher-risk MDS should be investigated to further promote patient-centered care.

Intravenous injection of bortezomib, melphalan and dexamethasone in refractory and relapsed multiple myeloma.

BACKGROUND: A combination of bortezomib (1.3 mg/m(2)), melphalan (5 mg/m(2)), and dexamethasone (40 mg) (BMD), with all three drugs given as a contemporary intravenous administration, was retrospectively evaluated. PATIENTS AND METHODS: Fifty previously treated (median 2 previous lines) patients with myeloma (33 relapsed and 17 refractory) were assessed. The first 19 patients were treated with a twice-a-week (days 1, 4, 8, 11, 'base' schedule) administration while, in the remaining 31 patients, the three drugs were administered once a week (days 1, 8, 15, 22, 'weekly' schedule). RESULTS: Side-effects were predictable and manageable, with prominent haematological toxicity, and a better toxic profile in 'weekly' schedule (36% versus 66% in 'base' schedule). The overall response rate was 62%. After median follow-up of 24.5 months (range 2.7-50 months), the median progression-free survival (PFS) was 21.6 with no difference between the two schedules and the median overall survival (OS) was 33.8 months. Independently from the adopted schedule, we found that also in a cohort of relapsed/refractory patients achieving at least partial remission improved PFS (35.2 versus 9 months) and OS (unreached median versus 18 months). CONCLUSION: Taken together, our observations suggest that BMD is an effective regimen in advanced myeloma patients with acceptable toxicity.

Prognostic value of CD34+ peak in peripheral blood during mobilization in intermediate-risk AML patients treated in first CR by autologous or allogeneic transplantation.

Ninety-six AML patients in 1st CR were evaluated for peak CD34+ cell levels in peripheral blood (PB) during PBSC mobilization and harvest. Distribution of CD34+ cell peaks was determined and cases were grouped on the basis of 50th and 75th percentile: group A, those having a CD34+ cell peak ≤70 × 10(9)/L (n=48); group B, those having a CD34+ cell peak between 70 and 183 × 10(9)/L (n=24); group C, those having a CD34+ cell peak >183 × 10(9)/L (n=24). Irrespective of post-remission treatment received, group A had a disease free survival (DFS) of 73%, group B a DFS of 51% and group C of 30% (P=0.0003). In intermediate cytogenetic risk patients, those treated by autologous transplantation had a DFS of 68, 33 and 14% in the groups A, B and C, respectively, (P=0.01) whereas after allogeneic transplantation DFS was 87% in group A+B vs 50% in group C (P=0.009). The peak of CD34+ cells in PB, was an independent predictor for DFS in multivariate analysis.

BRIT1/MCPH1 expression in chronic myeloid leukemia and its regulation of the G2/M checkpoint.

BRIT1 (BRCT-repeat inhibitor of hTERT expression), also known as microcephalin (MCPH1), is a crucial gene in the complex cellular machine that is devoted to DNA repair and acts as a regulator of both the intra-S and G2/M checkpoints. The most important role of BRIT1/MCPH1 in the regulation of cell cycle progression appears to be the G2/M checkpoint. The K562 and peripheral blood cells of chronic myeloid leukemia (CML) patients at diagnosis were found to downregulate BRIT1/MCPH1. However, we could not find any correlation between bcr/abl activity and the BRIT1/MCPH1 level. In order to study the genomic instability of CML cells, we evaluated the ability of these cells to arrest mitotic division after exposure to hydroxyurea, a known genotoxic agent. We showed that CML cells continue to proliferate without the activation of the G2/M cell cycle checkpoint arrest or of the apoptotic mechanism. This behavior may predispose the cells to accumulate genomic defects. In conclusion, we found that CML cells have a low BRIT1/MCPH1 level and show a defective G2/M arrest, confirming that these cells have a constitutive genomic instability.

Vascular endothelial growth factor polymorphisms in mantle cell lymphoma.

In this study, we determined the allele and genotype frequencies of vascular endothelial growth factor (VEGF) G+405C, C-460T, C+936T and C-2578A single nucleotide polymorphisms (SNPs) in 32 patients affected by mantle cell lymphoma (MCL) and 58 healthy controls. Real-time PCR combined with melting curve analysis was used for the determination of SNP alleles. A significant difference in the allele frequency of VEGFC-460T and C+936T SNPs in MCL and healthy cases was not observed. On the contrary, VEGF G+405C and C-2578A SNP allele distribution was significantly lower in the patient group than among normal controls (p = 0.014, p = 0.001). This observation suggests that further investigation is warranted, both in vitro and in a larger series of patients, to further examine the role of VEGF polymorphisms in the pathogenesis of MCL. In addition, the use of quantitative real-time PCR combined with a melting curve analysis method in the detection of the 4 VEGF SNPs may have the potential to replace older and more time-consuming PCR-RFLP methods and bears further investigation.

The efficacy of rituximab plus Hyper-CVAD regimen in mantle cell lymphoma is independent of FCgammaRIIIa and FCgammaRIIa polymorphisms.

Mantle cell lymphoma (MCL) accounts for 3-10% of all non-Hodgkin's lymphomas, with median overall survival not exceeding 3-4 years. Rituximab in combination with the Hyper-CVAD regimen appears the most promising regimen; thus, we adopted it as a first-line treatment strategy in a series of 24 patients. In addition to evaluation of clinical success of the regimen, we investigated a possible role of polymorphism in IgG Fc receptors, FCgammaRIIIa and FCgammaRIIa. The frequencies of FCgammaRIIIa-158 were as follows: V/V=4/24 (17%); V/F=16/24 (66%); F/F=4/24 (17%). Those of the FCgammaRIIa-131 polymorphism were H/H=11/24 (46%), H/R=9/24 (37%), R/R=4/24 (17%). The overall response rate was 62.5%, with 33% of complete responses (CRs) after four cycles of R-Hyper-CVAD. Two-year progression-free survival (PFS) was 78% for 158V/V patients vs 75% for cases carrying phenylalanine (p=0.88). When the FCgammaRIIa polymorphism was assessed, the 2-year PFS was 82% for 131H/H patients vs 75% for those carrying arginine (p=0.26). Eighty-three percent of cases achieved Polymerase Chain Reaction (PCR)-negativity: the progression rate was significantly influenced by the minimal residual disease clearance, with 12% progression in the subgroup of PCR-negative cases versus 67% progression in PCR-positive cases (p=0.008). The achievement of PCRnegativity was not significantly influenced by FCgammaR polymorphisms. Results confirm that rituximab plus Hyper-CVAD is an effective regimen for the induction of prolonged remission in patients with aggressive MCL and suggest that rituximab efficacy is independent of the FCgammaR polymorphisms.

Is endoscopic ultrasound clinically useful for follow-up of gastric lymphoma?

BACKGROUND: Endoscopic ultrasound (EUS) is considered the best technique for locoregional staging at diagnosis but its role in the follow-up of patients with gastric lymphoma after organ-conserving strategies has not been established. DESIGN AND METHODS: We retrospectively evaluated 23 patients with primary gastric lymphoma treated with a stomach-conservative approach. Sixteen of them were affected by MALT lymphoma and seven by diffuse large-B-cell lymphoma (DLBCL). Five patients were treated with Helicobacter pylori (HP) eradication therapy alone (omeprazole + amoxicillin + clarithromycin); eight patients received a treatment including HP eradication and chemotherapy and the remaining 10 patients were treated with chemotherapy alone. RESULTS: At the end of treatment, a complete remission was documented in 21 (91%) patients by endoscopy with biopsy (E-Bx) but in only seven (30%) patients by EUS. A total of 99 evaluations with both EUS and E-Bx were evaluated and we found concordance between the two methods in 33 occasions (33%) only. No significant difference on the percentage of concordance was recorded between MALT and DLBCL. After a median follow-up of 36.5 months we have not observed any relapse in 12 patients (six DLBCL and six MALT) with a persistent positive EUS but negative E-Bx. CONCLUSIONS: Although the length of follow-up cannot exclude late relapse, we think that in restaging and follow-up of gastric lymphoma, EUS seems not to be a reliable tool if it is abnormal and E-Bx still remains the gold standard. Therefore, after conventional conservative treatment, persistence of EUS abnormality with a negative histology should not be considered as a clinically relevant persistence of disease and should not be a reason for further treatment.

[Management of multiple myeloma]. / Terapia del mieloma multiplo.

Multiple myeloma (MM) remains an incurable malignancy, the median overal survival of patients receiving conventional chemotherapy being only 36-60 months. MGUS can evolve to MM in a percentage of 0.6-3% per year. The therapeutic management of multiple myeloma (MM) for the last several decades has mainly involved regimens based on use of glucocorticoids and cytotoxic chemotherapeutics. Melphaln and Prednisone (MP) are recognized as the classic treatment of MM. In patients candidate to bone marrow transplantation, VAD (Vincrisrine, Adriamicin, Dexamethasone) regimen is more indicated because it does not cause stem cell injury. High dose chemotherapy and and Autologous stem cells transplantation represent the best treatment for patients with MM who are younger than 65 years and free of severe comorbidities. Thalidomide alone or in combination with steroids has significant activity in multiple myeloma (MM). After the role of thalidomide in the management of patients with advanced or refractory MM had been established, many studies are evaluating the efficacy and toxicity of thalidomide as first-line therapy for patients with newly diagnosed disease. Recent studies have enhanced our understanding of disease pathogenesis and also provided the framework for a new treatment paradigm targeting the MM cell in its bone marrow microenvironment to overcome drug resistance and improve patient outcome. Clinical trials are confirming the remarkable activity and improved tolerability of some of the new agents identified through this paradigm.

Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis.

A high expression of Wilms' tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon. The aim of this study was to measure, by real-time polymerase chain reaction, levels of WT1 and MDR1 expression, in order to find a possible association between these genes, in a series of 50 newly diagnosed AML cases. Twenty-five percent of patients carried very high (>75 degrees percentile) MDR1- and 23.3%WT1-mRNA levels. Interestingly, high levels of WT1 were significantly correlated with correspondent high levels of MDR1 gene. Nevertheless, the co-expression of these genes did not significantly influence the complete response rate to the induction therapy. Reported data confirm the existence of a co-expression of WT1 and MDR1 genes even in vivo; this may be relevant because one consequence could be the positive selection by chemotherapeutic regimens of cells with higher MDR1 levels already present before treatment. Thus, the association between these genes could suggest avoiding the use of drugs involved in the multidrug resistance (MDR) phenomenon in patients carrying high levels of WT1 at diagnosis.

Quantitative molecular evaluation in autotransplant programs for follicular lymphoma: efficacy of in vivo purging by Rituximab.

The main aim of this paper was to compare results of Genescan and real-time PCR methods in order to detect contamination in harvests from patients with follicular lymphoma. The secondary goal was to evaluate the efficacy of Rituximab as an in vivo purging agent. A total of 23 patients had been treated with CHOP followed by either high-dose therapy (12 patients) or high-dose plus Rituximab (11 patients), both followed by autologous transplantation. Results show that 86% of harvests from patients treated with Rituximab were PCR-negative compared to 14.3% from controls. Real-time PCR was more sensitive than Genescan PCR; quantitative analysis revealed a correlation between the amount of contamination in the harvests and relapse after transplantation. Whereas all patients reinfused with negative aphereses achieved complete remission and showed a significantly better 5-year PFS (100%) compared to those reinfused with contaminated samples (41%), a very low amount of contamination does not appear to negatively affect outcome, suggesting that determination of a cutoff in the contamination level of harvests could be useful. Results suggest that real-time PCR is superior to Genescan PCR to select transplantable harvests and confirm the ability of Rituximab as an in vivo purging tool for follicular lymphoma.

Elevated vascular endothelial growth factor (VEGF) serum levels in idiopathic myelofibrosis.

An increase of angiogenesis has been shown in idiopathic myelofibrosis with myeloid metaplasia (MMM) by microvessel density count method but evaluation of circulating angiogenic factors is still incomplete. In 31 patients affected by MMM and in 12 healthy subjects we evaluated the serum levels of VEGF (vascular endothelial growth factor) and correlated VEGF with clinical and laboratory features of disease. We found that MMM patients had circulating VEGF concentrations much higher than controls (Median 1208 ng/ml vs 138 ng/ml, P < 0.0001). No correlation was found between VEGF and Hb, WBC, PLT, LDH, creatinine, bone marrow cellularity, fibrosis, splenomegaly, hepatomegaly, and therapy. However, in the subgroup of patients with a normal or low VEGF concentration, a direct correlation between VEGF and platelet count (r = 0.90, P = 0.002) was detected. Moreover, patients with a platelet count < 300 x 10(9)/l had VEGF serum levels lower than patients with a higher PLT count (median VEGF 864 vs 1557 pg/ml, P = 0.001). In six patients and in eight controls we also had the opportunity to measure VEGF in the plasma and we calculated that VEGF concentration was much higher in platelet-rich than in platelet-poor plasma and that platetets of MMM patients contained four times more VEGF than those of healthy controls. These results indicate that VEGF is overproduced in MMM, thus confirming an increased angiogenic activity. Platelets are probably a major source of VEGF in MMM but not the only one.

ABL1 methylation in Ph-positive ALL is exclusively associated with the P210 form of BCR-ABL.

In human Ph-positive leukemia there is a clear association of different forms of the BCR-ABL oncogene with distinct types of leukemia. The P190 form of BCR-ABL is rarely observed in chronic myeloid leukemia (CML) but is present in 50% of Ph-positive acute lymphoblastic leukemia (ALL). In contrast, the P210 form is observed both in CML and 50% of Ph-positive ALL. Methylation of the proximal promoter of the ABL1 gene has been shown to be a nearly universal event associated with clinical progression of CML. This raises the question of whether methylation of the ABL1 promoter is an epigenetic modification also associated with Ph-positive ALL. To study this issue, we used methylation-specific PCR and bisulfite sequencing to determine the methylation status of the ABL1 promoter in 18 Ph-positive ALL samples. We report here that gene-specific ABL1 promoter methylation is associated mainly with the P210 form of BCR-ABL and not the P190 form. While six out of the seven P210-positive ALL samples had ABL1 promoter methylation, none of the 11 P190-positive ALL samples demonstrated ABL1 promoter methylation. In addition, we estimated the extent and relative abundance of ABL1 promoter methylation in several Ph-positive ALL samples and compared it to the methylation pattern in chronic, accelerated and blastic crisis phases of CML. We put forth a model that correlates the different types of leukemias with the different levels of ABL1 promoter methylation.

Angiogenesis in chronic myeloproliferative diseases.

Increased angiogenic activity has been demonstrated in myelofibrosis with myeloid metaplasia (MMM), chronic myeloid leukemia (CML), and essential thrombocythemia (ET) by both bone marrow microvessel density evaluation and measurement of circulating angiogenic factors. MMM is probably the disease with the more pronounced angiogenesis among myeloproliferative disorders but the significance of this finding remains speculative since the angiogenic activity is not correlated with any of the clinical and laboratory features of the disease. Circulating serum levels of angiogenic factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were found increased in MMM, CML and ET but the frequent thrombocytosis that accompanies these diseases could limit the interpretation of these data since platelets and megakaryocytes may be considered a major source at least for VEGF. However, CML patients treated with interferon were found to have lower VEGF and HGF levels than untreated or hydroxyurea-treated patients, thus suggesting a possible antiangiogenic mechanism of this drug. In addition, preliminary experiences with the antiangiogenic drug thalidomide have shown therapeutic activity in some myeloproliferative disorders.

In vitro apoptotic response of freshly isolated chronic myeloid leukemia cells to all-trans retinoic acid and cytosine arabinoside.

Chronic myeloid leukemia (CML) is a hematological malignancy resulting from clonal expansion and massive accumulation of leukemic myeloid cells that retain differentiation and maturation capacity. Since CML cell accumulation has been related to apoptosis inhibition by the product of the BCR-ABL gene, attempts to eradicate leukemic cells would require therapeutic drugs able to overcome this inherent resistance. Here, we investigated in vitro the apoptotic effect of all-trans retinoic acid (ATRA) and cytosine arabinoside (ARA-C), employed alone, in combination or in sequence, on freshly isolated cells from 10 patients with chronic-phase CML. Our cell cultures showed that both ATRA and ARA-C were able to induce apoptosis in CML cells, even if ARA-C resulted more effective than ATRA. The combined use of ATRA and ARA-C seemed to have only an additive effect while the sequential use did not show any advantage. These in vitro observations indicate that ATRA and ARA-C may be effective in reducing CML cells through apoptosis induction, suggesting that it could be worthwhile to examine ATRA and ARA-C combinations in the therapy of CML.

The tryphostin AG17 induces apoptosis and inhibition of cdk2 activity in a lymphoma cell line that overexpresses bcl-2.

Tyrphostins are low molecular weight compounds that specifically inhibit protein tyrosine kinases. We studied the effects of tyrphostins on OCI-Ly8, a cell line derived from a patient with immunoblastic lymphoma that carries the t(14;18) translocation and overexpresses the B-cell lymphoma/leukemia-2 gene (bcl-2). To test the possibility that tyrphostins induce apoptosis in these cells, overcoming the protection rendered by bcl-2, we screened 16 tyrphostins representing different families at a concentration of 0.5-50 microM. We found that AG17 was the most potent in this regard. Cell cycle analysis demonstrated that AG17 induces arrest at the G1 phase followed by apoptosis with general reduction of the intracellular level of tyrosine-phosphorylated proteins. To further elucidate the mechanism of action of AG17, we investigated its effect on some of the key proteins that regulate the cell cycle. Bcl-2 and cdk2 protein levels were not altered with AG17, whereas cdk2 kinase activity, as well as p21 and p16 protein levels, were reduced markedly. These results suggest that the target of AG17 is inactivation of cdk2. Because lymphoma cells with the t(14;18) translocation and bcl-2 overexpression are resistant to chemotherapy, novel drugs selectively able to induce apoptosis in these cells could offer a new approach to the treatment of lymphoma patients.