Phase I study of cloretazine (VNP40101M), a novel sulfonylhydrazine alkylating agent, combined with cytarabine in patients with refractory leukemia.

PURPOSE: Cloretazine (VNP40101M) is a novel sulfonylhydrazine alkylating agent with significant antileukemia activity. A phase I study of cloretazine combined with cytarabine (1-beta-d-arabinofuranosylcytosine, ara-C) was conducted in patients with refractory disease. DESIGN: Ara-C was given i.v. at a fixed dose of 1.5 gm/m(2)/d by continuous infusion for 4 days (patients ages <65 years at time of diagnosis) or 3 days (patients ages > or =65 years). Cloretazine was given i.v. over 15 to 60 minutes on day 2 at a starting dose of 200 mg/m(2), with escalation in 100 mg/m(2) increments in cohorts of three to six patients until a maximum tolerated dose was established. The DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) was measured at baseline. RESULTS: Forty patients, including 32 with acute myeloid leukemia, received 47 courses of treatment. Complete responses were seen at cloretazine dose levels of > or =400 mg/m(2) in 10 of 37 (27%) evaluable patients, and in this patient subset, AGT activity was significantly lower in patients that responded to treatment than in patients who did not (P < or = 0.027). Dose-limiting toxicities (gastrointestinal and myelosuppression) were seen with 500 and 600 mg/m(2) of cloretazine combined with the 4-day ara-C schedule but not seen with the 3-day schedule. CONCLUSION: The recommended cloretazine dose schedule for future studies is 600 mg/m(2) combined with 1.5 gm/m(2)/d continuous infusion of ara-C for 3 days. The cloretazine and ara-C regimen has significant antileukemic activity. AGT activity may be a predictor of response to cloretazine.     

p16INK4A and p15INK4B gene deletions in primary leukemias

The 9p21 locus has been deleted at a high frequency in a wide variety of tumors. Recently, two genes, p16INK4A and p15INK4B (also called MTS1 and MTS2), have been localized in close proximity at the 9p21 locus, encoding cyclin-dependent kinases 4/6 inhibitors of relative molecular mass 16 kD and 15 kD, respectively and also found to be deleted at a high frequency in tumor cell lines. We analyzed p16INK4A and p15INK4B genes in 178 cases of primary leukemias including 81 cases of chronic lymphocytic leukemia (CLL), seven of hairy cell leukemia (HCL), seven of chronic myelogenous leukemia (CML), 43 of acute myelogenous leukemia (AML), 27 of acute lymphoblastic leukemia (ALL), and 13 of myelodysplastic syndrome (MDS) by Southern blot analyses. The ALL cases showed a relatively high frequency of homozygous deletions (22%, 6 of 27) at the p16INK4A gene locus. Interestingly, of the six cases with p16INK4A homozygous deletions, only three showed homozygous deletions at the p15INK4B gene. In 81 CLL patients, we detected one homozygous and five heterozygous deletions at both the p16INK4A and p15INK4B genes and two heterozygous deletions at the p16INK4A gene alone. Deletion of these two genes in AML cases is relatively low (9%). We did not detect deletions in any of the MDS, HCL, and CML cases examined. Sequence analyses of p16INK4A gene of six CLL cases with heterozygous deletion at this locus showed a 27-bp deletion at the splice acceptor site of intron 1 in one case and changes in the coding sequence in three other cases. The data presented in this report showed that (1) p16INK4A and p15INK4B genes are preferentially deleted homozygously in ALL and heterozygously in CLL cases with frequent mutation in the second allele, and (2) p16INK4A gene appears to be more frequently deleted than p15INK4B gene.     

Developmental switching of messenger RNA expression from the human alpha-globin cluster: fetal/adult pattern of theta-globin gene expression.

The alpha-globin gene cluster contains four functional globin genes, zeta, alpha 2, alpha 1, and theta. The developmental regulation of the embryonic zeta and fetal/adult alpha 2- and alpha 1-globin genes is well characterized at the level of protein synthesis. The developmental pattern of the theta-globin gene is not well characterized due to the inability to detect its encoded protein. Direct analysis of the globin switching at the steady-state messenger RNA (mRNA) level has been hampered by the difficulty in obtaining quantities of embryonic and early fetal mRNA sufficient for analysis. We analyzed the relative levels of the steady-state zeta-, alpha-, and theta-globin mRNAs in yolk sac in 5-, 6-, 7-, and 8-week postconception embryonic liver, and in cord and adult blood reticulocytes. We show that the switch in the alpha-globin gene cluster from the embryonic to fetal/adult pattern of expression begins at 5 to 6 weeks of gestation. Both the theta- and alpha-globin genes show similar patterns of developmental control that are reciprocal to zeta. alpha-globin RNA is barely detectable or undetectable at 5 weeks, and increases in the 6- to 8-week period, while theta-globin mRNA shows a parallel increase at 5 to 8 weeks postconception and is expressed in cord blood and adult reticulocytes. These data show that the theta-globin gene represents a fetal/adult gene, albeit expressed at a low level.     

Human embryonic zeta-globin chain expression in deletional alpha-thalassemias.

zeta-Globin chain expression in carriers of a number of deletional alpha-thalassemias is investigated by radioimmunoassay. In a few cases, zeta-globin mRNAs are also studied. zeta-Globin chains are detected in (--SEA/), (--MED/), and (--SPAN/) deletions, but not in six other deletional mutations. These results suggest that the DNA element capable of suppressing zeta-globin expression in adult erythroid cells is present within the (--SPAN/) deletion, while the DNA fragment between the 5' breakpoints of the (--SA/) and the (--SEA/) deletions may contain sequences necessary for augmenting zeta-globin expression in adult erythroid cells. Furthermore, zeta-globin chains are shown by an immunocytologic technique to be present in all circulating erythrocytes in carriers of the (--SEA/) and (--MED/) deletions. This simple immunocytologic test is highly sensitive and specific to detect adult carriers of either the (--SEA/) or (--MED/) deletions, and can be used for the detection of couples at risk of pregnancies involving fetuses with homozygous alpha-thalassemia.     

Somatic mutations of signaling genes in non-small-cell lung cancer

Lung cancer is the leading cause of cancer-related deaths, with non-small-cell lung cancer (NSCLC) accounting for approximately 85% of cases. A significant proportion of NSCLC cases are not diagnosed until a late stage, when aggressive treatments are required but often prolong survival only modestly. Recent advances in molecular characterization of NSCLC have enabled identification of numerous cell growth and proliferation pathways that are disrupted in these tumors. This knowledge has provided insight into the mechanisms of tumor development in various histologic subtypes of NSCLC and has pointed the way toward targeted treatment strategies. In this review, we highlight literature findings of somatic mutations in genes involved in cell growth and proliferation that are commonly found in the various subtypes of NSCLC, and we discuss how these findings may relate to treatment strategies.     

A prognostic model for survival in chronic lymphocytic leukaemia based on p53 expression

As the abnormal expression of p53 protein is prognostically significant in some human cancers, its significance in patients with B-cell chronic lymphocytic leukaemia (CLL) was assessed. Two investigators evaluated the percentage of bone marrow mononuclear cells that stained for p53, using biopsies stained with anti-p53 monoclonal antibody (DO-7), and graded the degree of staining (0, +, ++, +++). Samples from a cohort of 90 patients with CLL were studied (median age 60 years, range 30-89 years; 57 patients were (63%) previously untreated, 22 patients (24%) had received one or two prior regimens, 11 patients had received (12%) three to seven regimens. The overall percentage of cells positive for p53 staining was a median of 43 (range 1-88). No investigator effect was detected either in overall percentage cells rated p53 positive or on the degree of staining (Pearson's correlation coefficient 0.980, P-value < 0.001). A Cox proportional hazards model showed that the percentage of ++ and +++ p53-positive cells correlated with various prognostic factors in CLL (P < 0.0001). A multivariate model incorporating prior therapy, Rai stage, beta2 microglobulin (beta2M) and p53 expression showed that only the percentage of p53-positive cells and beta2M were predictive of survival, and enabled the development of a highly predictive model of survival based on these two parameters.     

The clinical and diagnostic relevance of CD23 expression in the chronic lymphoproliferative disease

BACKGROUND: CD23 antigen is a cell surface protein considered important in the differentiation of chronic lymphocytic leukemia (CLL) from other lymphoid leukemias. METHODS: To better clarify CD23 role as a diagnostic tool, the authors retrospectively evaluated clinical and laboratory features of 372 patients who were referred to M.D. Anderson Cancer Center with a diagnosis of CLL or B-cell chronic lymphoproliferative disease. RESULTS: Most of the patients (91%) were CD19+/CD5+. Only 6% of these CD19+/CD5+ patients were CD23-. Overall, CD23- patients had the worse prognostic features compared with CD23+ cases, including anemia (P = 0.03), massive splenomegaly (P = 0.000), high lactate dehydrogenase (P = 0.007), high beta2-microglobulin (P = 0.006), older age (P = 0.001), and male gender (P = 0.02). Surface immunoglobulin expression was moderate/strong in 19 (82%) patients, and FMC-7 was positive in 22 (96%) patients. None of the 13 patients tested for CD10 expressed the antigen. Based on morphology, of the CD23, 16 (70%) were diagnosed with mantle cell leukemia (MCL) was diagnosed in 16 (70%) CD23- patients, 3 (13%) with splenic marginal-zone leukemia, 3 (13%) with prolymphocytic leukemia (PLL) or PLL/CLL, and 1 (4%) with CLL. No cyclin D1 protein expression was noted by Western blot analysis in the one case that showed typical CLL morphology, and this patient did not require therapy. On the whole, the survival rate of CD23- patients was significantly worse than that of patients with CD23+. In contrast, 15 of 32 (49%) CD19+/CD5- patients were CD23-. CD23 negativity in this group was not associated with distinct clinical features or outcome. Eleven (73%) of these patients were classified as having splenic marginal-zone lymphoma and 4 as having follicular lymphoma. CONCLUSIONS: These data indicate that CD23 negativity is rare in typical B-cell CLL, and CD23 negativity in patients with CD19+/CD5+ is suggestive of mantle cell leukemia a more aggressive disease with poor response to conventional therapy in which newer chemotherapy regimens such as hyper-CVAD may be more effective.     

Less apoptosis in patients with 5q-syndrome than in patients with refractory anemia

The WHO classification of hematological malignancies includes 5q-syndrome as a separate category within myelodysplastic syndromes (MDS). Clinically, patients with 5q-syndrome have a milder disease than patients with other MDS. The basis for this difference is not known. Identifying 5q-syndrome can be difficult because some of its morphologic and cytogenetic features are similar to those of other MDS. We compared apoptosis between 5q-syndrome and other refractory anemias. We found lower levels of apoptosis in 5q-syndrome as detected by less disruption of mitochondrial potential (P=0.008) and decreased annexin V positivity (P=0.01). Our results suggest that lower apoptosis in 5q-syndrome may explain the milder clinical course of the disease and distinguish 5q-syndrome from other MDS.