Carlos J. Carrera

Lasting remissions in hairy-cell leukemia induced by a single infusion of 2-chlorodeoxyadenosine.

2-Chlorodeoxyadenosine is a simple purine nucleoside that has previously been shown to be effective in the treatment of low-grade malignant disorders of lymphoid tissue, including chronic lymphocytic leukemia and non-Hodgkins lymphoma. Because of these encouraging results, we treated 12 patients with another low-grade B-cell neoplasm, hairy-cell leukemia. The patients received 2-chlorodeoxyadenosine (0.1 mg per kilogram of body weight per day) by continuous infusion for seven days. All the patients responded to treatment. Eleven had complete remissions characterized by the normalization of peripheral blood and bone marrow and disappearance of tumor masses. The longest remission has been 3.8 years. None of the patients have relapsed, and the median duration of remission has been 15.5 months. No serious toxic reactions occurred as a result of 2-chlorodeoxyadenosine therapy. These results suggest that 2-chlorodeoxyadenosine may be the most effective therapy available for hairy-cell leukemia. The administration of 2-chlorodeoxyadenosine resulted in a higher rate of complete remission than is observed with interferon alfa, and it required no maintenance therapy. Its toxicity may be lower than that of deoxycoformycin, and the responses were achieved with single courses of treatment.

2-Chlorodeoxyadenosine treatment of low-grade lymphomas.

PURPOSE Because of the need to identify effective new agents in the treatment of non-Hodgkins lymphoma and because of the high activity of the purine analog 2-chlorodeoxyadenosine (2-CdA) against chronic lymphocytic leukemia and hairy cell leukemia, a phase II trial of 2-CdA was initiated in patients with low-grade lymphocytic lymphomas. PATIENTS AND METHODS Forty patients with low-grade lymphocytic lymphomas including diffuse small lymphocytic, follicular small-cleaved, and follicular mixed histologies were enrolled onto the study. Conventional therapies had failed in all patients, and six patients had lymph node biopsies showing evidence of histologic evolution to a higher-grade lymphoma. A total of 107 courses of 2-CdA were administered. There were 27 males and 13 females. The median age was 59 years (range, 37 to 80 years). Patients had received a median of three prior therapies (range, one to six therapies). RESULTS An overall response rate of 43% was achieved, with eight patients experiencing complete responses (CRs) and nine patients experiencing partial responses (PRs). The duration of responses ranged from 1 to greater than 33 months without maintenance therapy (median duration of response, 5 months). Histology and prior therapy history did not seem to correlate with responses. Significant toxicity was limited to bone marrow suppression; 18% of patients developed neutropenia, and 30% developed thrombocytopenia. CONCLUSIONS This phase II trial demonstrates that 2-CdA is an effective antilymphocyte, antineoplastic agent with significant activity as a single agent in patients with recurrent or refractory low-grade lymphocytic lymphoma. Responses were achieved with an acceptable toxicity profile. Further trials of this agent in previously untreated patients and in combination regimens are indicated and will be developed.

Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than p16INK4A (CDKN2) homozygous deletions in primary non-small cell lung cancers (NSCLC).

Homozygous deletions of the tumor suppressor gene p16INK4A and deficiency of methylthioadenosine phosphorylase (MTAP), both located on chromosome 9p21, have been independently reported in non-small cell lung cancer (NSCLC). To determine the frequency of co-deletion of these two genes, we investigated 50 samples of primary NSCLC using a quantitative PCR-ELISA. All specimens were fixed in formalin, paraffin embedded and stored until assayed. Histologic subtypes included 25 adenocarcinomas (50%), 21 squamous cell carcinomas (42%) and four large cell carcinomas (8%). Homozygous deletions of MTAP exon 8 could be detected in 19 of 50 NSCLC samples (38%). Adenocarcinoma (11 of 25, 44%) showed a higher deletion frequency than squamous cell carcinoma (six of 21, 29%). In contrast, homozygous p16INK4A deletions were detected in only nine of 50 (18%) samples using specific primers for p16INK4A exon 1α. No difference between the histological subtypes and p16INK4A deletion frequency was observed. We further investigated the ten samples with MTAP deletions but intact p16INK4A exon 1α with primers specific for p16INK4A exon 3, the exon nearest to MTAP exon 8. Interestingly, none of the ten samples had deletion of the p16INK4A exon 3 coding region. Fine mapping analysis performed in ten samples showed a frequent breakpoint between MTAP exon 4 and exon 5. In addition, p16 protein expression could not be detected in five out of six samples with intact p16 but deleted MTAP locus. These data show a high frequency of homozygous MTAP deletions in NSCLC which is associated with detectable co-deletion of p16INK4A in only half of the cases. This result suggests the existence either of another tumor suppressor gene telomeric of p16INK4A or of deletions involving 3′-untranslated (3′-UTR) regulatory regions of p16INK4A that can interfere with its expression or function.

2-Chlorodeoxyadenosine dose escalation in nonhematologic malignancies.

PURPOSE We performed a dose-escalation study of 2-chlorodeoxyadenosine (2-CdA) in solid tumors to determine the maximum-tolerated dose (MTD) and define its toxicity profile at higher doses. PATIENTS AND METHODS Twenty-one patients, seven with malignant astrocytoma, twelve with metastatic melanoma, and two with metastatic hypernephroma, were enrolled onto the study. Patients were entered onto cohorts that received 0.10, 0.15, or 0.20 mg/kg/d of 2-CdA by continuous intravenous infusion for 7 days every 28 days. 2-CdA levels were determined by radioimmunoassay. In tumor tissue samples, deoxycytidine kinase (dCK) levels were measured by both enzyme activity and immunoreactive protein analysis. RESULTS Of seven patients treated with 2-CdA at 0.1 mg/kg/d, one experienced grade 3 or 4 myelotoxicity. Of 11 patients treated at 0.15 mg/kg/d, four experienced myelotoxicity, two after a single course of 2-CdA. All three patients who received 2-CdA at 0.2 mg/kg/d experienced myelosuppression. Neurologic events occurred in two patients, both with malignant melanoma. Two of seven patients (28.6%) with astrocytomas obtained partial responses with a median duration of 8 months. 2-CdA penetrated the blood-brain barrier. An association was found between dCK levels as measured by enzymatic activity and immunoreactive proteins, but this did not correlate with 2-CdA tumor responsiveness. CONCLUSION The MTD for 2-CdA delivered as a 7-day intravenous infusion in patients with nonhematologic malignancies was determined to be 0.1 mg/kg/d, the same as the MTD for patients with hematologic malignancies. There was no clinical correlation with dCK expression and response to 2-CdA. The responses noted in patients with malignant astrocytoma warrant further phase II study.

A methylthioadenosine phosphorylase (MTAP) fusion transcript identifies a new gene on chromosome 9p21 that is frequently deleted in cancer

Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines, and are also common in primary gliomas, lung cancers, and leukemias. Moving from the centromere to the telomere, this complex region encodes the tumor suppressor genes p15INK4B (CDKN2B), p14ARF, p16INK4A (CDKN2A), and the housekeeping gene methylthioadenosine phosphorylase (MTAP). However, not all chromosome 9p21 deletions in tumors include these tumor suppressor genes. Here we describe the partial sequence and the exact localization of a new gene on chromosome 9p21 centromeric of p15INK4B, that formed an in frame fusion transcript with MTAP in a glioma xenograft, and that is homozygously deleted in various malignant cell lines. Northern blot revealed corresponding 1.5 kb transcript in non-deleted cell lines as well as in normal lymphocytes. Using a RNA master blot membrane including 50 different tissues, we could show that this new transcript is expressed in all tissues of the adult but not or only at very low levels in most of the fetal tissues tested. The expression pattern is similar to that of p16INK4A. The localization as well as the deletion pattern makes this transcript a candidate for a new tumor suppressor gene.

Marrow Suppression Produced by Repeated Doses of Cladribine

2-Chlorodeoxyadenosine (cladribine, Leustatin) is being used extensively in the treatment of hematologic malignancies, but relatively little is known regarding its toxicity to the normal marrow. Long-term serial hematologic observations have been made on 29 patients with multiple sclerosis undergoing experimental therapy with monthly courses of cladribine, each of which consisted of 0.087-0.1 mg/kg per day for 7 days. The characteristic hematologic responses of the patients consisted of acute transient monocytopenia, prolonged, profound lymphopenia especially of CD4-positive cells, and modest lowering of the granulocyte count and hemoglobin with development of long-lasting macrocytosis. Two patients developed severe aplastic anemia, requiring transfusion both of red cells and platelets. One of these had previously received extensive therapy with chlorambucil, while the other had received carbamazepine (Tegretol) and was ingesting phenytoin (Dilantin) at the time of cladribine therapy. Both patients recovered after several months of marrow suppression.

Hairy cell leukemia: New understanding of biology and treatment

The term Leukemic reticuloendotheliosis, later to be known as hairy cell leukemia (HCL), was first applied by Ewald [1] to a patient with a fulminating, fatal disorder characterized by the circulation of large numbers of abnormal cells in the blood. Although his publication is sometimes cited as the first description of HCL [2], in retrospect it seems more likely that he was actually observing a patient with a form of acute myelogenous leukemia.

17 PROFOUND TOXICITY OF DEOXYADENOSINE (dAdo) AND 2-CHLORODEOXYADENOSINE (CdA) TOWARD HUMAN MONOCYTES IN VITRO AND IN VIVO

dAdo is known to be toxic to both proliferating and resting lymphocytes that lack adenosine deaminase (ADA) activity. We now show that human monocytes are also highly sensitive in vitro to nM concentrations of dAdo plus the ADA inhibitor deoxycoformycin, and to the ADA-res istant analog CdA. The dose- and time-dependent toxicity of dAdo or CdA to monocytes is blocked by deoxycytidine, implicating deoxycytidine kinase in the formation of toxic dAdo or CdA nucleotides. Monocytes exposed to dAdo plus deoxycoformycin, or to CdA accumulate massive DNA damage detectable within 1 hour. The accumulation of DNA strand breaks in lymphocytes stimulates the lethal consumption of NAD and ATP for poly(ADP-ribose) synthesis. However, monocytes lack the poly(ADP-ribose) polymerase enzyme and therefore show no significant NAD or ATP depletion until cell viability declines (12 hr). The DNA damage in monocytes exposed to CdA is associated with a decrease in protein synthesis in vitro, and with inhibition of IL-6 secretion. The selective toxicity of CdA to monocytes was confirmed by in vivo studies. Thus, the blood monocyte counts, but not the neutrophil counts, fell to 0 in one week in nearly all patients receiving CdA infusion chemotherapy for cutaneous lymphoma. These results show that dAdo and CdA cause DNA strand break formation and inhibit protein synthesis in human monocytes in vitro, and cause profound monocytopenia in vivo. These compounds may have potential use in the therapy of immune disorders associated with monocyte/macrophage activation.

18 POTENT ACTIVITY OF 2-CHLORODEOXYADENOSINE IN CHRONIC LYMPHOCYTIC LEUKEMIA, HAIRY CELL LEUKEMIA, AND AUTOIMMUNE HEMOLYTIC ANEMIA

Unlike other nucleoside anti-metabolites, 2-chlorodeoxyadenosine is selectively toxic at nanomolar concentrations to human lymphocytes and monocytes. In susceptible cells, the drug causes a dose- and time-dependent accumulation of DNA strand breaks, with resultant activation of poly(ADP-ribose) polymerase. Furthermore, the actions of 2-chlorodeoxyadenosine are entirely independent of replicative DNA synthesis. For this reason, we reasoned that 2-chlorodeoxyadenosine would represent a useful agent for the treatment of slowly replicating lymphoid malignancies, and for the therapy of chronic autoimmune diseases. In the present study, 2-chlorodeoxyadenosine was administered to 18 patients wich advanced chronic lymphocytic leukemia, 4 of whom had concurrent autoimmune hemolytic anemia. An overall response rate of 56% was achieved. Only minor and reversible bone marrow suppression occurred during treatment, indicating a high degree of lymphocyte selectivity. Moreover, 3 of the 4 patients with autoimmune hemolytic anemia had complete resolution of hemolysis. Three patients with hairy cell leukemia also received 2-chlorodeoxyadenosine therapy. Two of the patients achieved clinical remission after one course of the drug. These results demonstrate that 2-chlorodeoxyadenosine is a safe and potent anti-lymphocyte and immunosuppressive agent. Further trials of the drug in autoimmune and lymphoproliferative diseases are warranted.

19 2-HALO-2|[prime]|,3|[prime]|-DIDEOXYADENOSINES: METABOLICALLY STABLE DIDEOXYNUCLEOSIDES WITH ACTIVITY AGAINST THE HUMAN IMMUNODEFICIENCY VIRUS (HIV)

2′,3′-dideoxyadenosine (ddA) has activity against the human immunodeficiency virus-1 (HIV), but is rapidly catabolized by human T cells, even when adenosine deaminase is inhibited by deoxycoformycin. To overcome this problem, we developed a simple method to synthesize the 2-fluoro-, 2-chloro-, and 2-bromo-derivatives of ddA. The isolated 2-halo-ddA derivatives were not deaminated significantly by cultured T lymphoblasts, which converted the dideoxynucleosides to the respective 5′-monophosphate, 5′- diphosphate, and 5′- triphosphate metabolites. At concentrations lower than those producing cytotoxicity in uninfected cells (3-10 μM), the 2-halo-ddA derivatives inhibited the cytopathic effects of HIV toward T lymphoblasts, and retarded viral replication. Experiments with a deoxycytidine kinase deficient mutant CEM T cell line showed that this enzyme was necessary for the phosphorylation and anti-HIV activity of the 2-halo-ddA derivatives. Thus, the 2-halo-ddA congeners, in contrast to ddA itself, are not degraded by T lymphocytes, and represent promising compounds for in vivo chemotherapy of HIV infection.