Alan Saven

Gaucher Disease: Clinical, Laboratory, Radiologic, and Genetic Features of 53 Patients

We have reviewed our experiences with the clinical, laboratory, radiologic, and genetic features of 53 patients with Gaucher disease. Most were evaluated during early adult life, with a mean age of 33 years. Our patients were evaluated in a referral center, and therefore the data need to be interpreted with caution when applied to the general patient population, which includes a greater proportion of very mild cases. Thirty-nine patients were Ashkenazi Jews, 13 were non-Jewish and 1 was half-Jewish. The most common presenting symptom was bleeding related to splenomegaly and thrombocytopenia. The chronic symptoms, evaluated an average of 20 years after the diagnosis had been established, were mainly skeletal. Splenectomy had been performed in 43% of our patients and there was no evidence that this procedure accelerated the progression of liver and bone involvement. DNA from the patients was examined for 20 different mutations. The association between the 1226G/1226G genotype and a milder clinical course, and between the 1226G/84GG and 1226/1448C genotypes with more severe clinical manifestations, was confirmed. Repeated follow-up examinations in 29 patients revealed that in the majority of the patients, progression of the disease occurs during childhood, adolescence, or early adulthood with a marked tendency for stabilization thereafter. This observation suggests that Gaucher disease in most of the patients is not a relentless progressive disorder but a rather stable disorder during adulthood. The indications for the newly introduced intravenous enzyme replacement therapy as well as of future experimental treatments should be examined in the light of the natural history of the disease.

Activated integrin αvβ3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells

Expression of adhesion receptor integrin αvβ3 in an activated functional form strongly promotes metastasis in human breast cancer cells. Here, we report that αvβ3 cooperates with matrix metalloproteinase type 9 (MMP-9) in breast cancer cell migration. This cooperation is regulated by the activation state of the integrin. Expression of activated αvβ3 in metastatic variants of MDA-MB 435 human breast cancer cells and primary metastatic cells from breast cancer patients strongly enhanced migration toward vitronectin and fibrinogen. This enhancement was mediated by a soluble factor produced by breast cancer cells expressing activated αvβ3. When transferred, this factor also up-regulated αvβ3-dependent migration of breast cancer cells that express the nonactivated integrin. The factor was identified as metalloproteinase MMP-9. Whereas all tested breast cancer cell variants produced latent MMP-9, only those with activated αvβ3 produced the mature form of this metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either form of MMP-2, enhanced αvβ3-dependent breast cancer cell migration. The migratory response was inhibited by tissue inhibitors of metalloproteinase or when MMP-9 was depleted from the inducing supernatants. The results indicate a causal relationship between the expression of activated integrin αvβ3 and production of enzymatically active MMP-9 in metastatic breast cancer cells. These molecules cooperate to enhance breast cancer cell migration toward specific matrix proteins, and this may contribute to the strongly enhanced metastatic capacity of breast cancer cells that express activated αvβ3.

Extended Follow-Up of Patients With Hairy Cell Leukemia After Treatment With Cladribine

PURPOSE Hairy cell leukemia (HCL) is an uncommon, indolent, chronic B-cell lymphoproliferative disorder involving the marrow and spleen. Therapy for HCL includes splenectomy, interferon alfa-2a and alfa-2b, pentostatin, and cladribine. The purpose of this article was to report the extended follow-up of HCL patients treated with cladribine. PATIENTS AND METHODS Two hundred nine patients with HCL who were treated with cladribine had at least 7 years of follow-up. A course of cladribine constituted a 7-day continuous intravenous infusion at a dose of 0.1 mg/kg/d. RESULTS Of the 207 assessable patients who had at least 7 years of follow-up, 196 (95%) achieved a complete response (CR) and 11 (5%) achieved a partial response (PR) after a single course of cladribine (overall response rate, 100%). The median first-response duration for all responders was 98 months. Seventy-six patients (37%) experienced relapse after their first course of cladribine. The median time to first relapse for all responders was 42 months. Time to treatment failure of CRs compared with PRs was statistically significant (P <.0005). The overall survival rate was 97% recorded at 108 months. Forty-seven patients developed 58 second malignancies. The observed-to-expected ratio for second malignancies was 2.03 (95% confidence interval, 1.49 to 2.71). CONCLUSION These results confirm previous observations that single courses of cladribine administered to patients with HCL induce high response rates, the majority of which are CRs. Most patients enjoy long-lasting complete remissions, and those patients who experience relapse can be successfully re-treated with cladribine.

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.

In vivo targeting of B-cell lymphoma with glycan ligands of CD22.

Antibody-mediated cell depletion therapy has proven to provide significant clinical benefit in treatment of lymphomas and leukemias, driving the development of improved therapies with novel mechanisms of cell killing. A current clinical target for B-cell lymphoma is CD22, a B-cell-specific member of the sialic acid binding Ig-like lectin (siglec) family that recognizes alpha2-6-linked sialylated glycans as ligands. Here, we describe a novel approach for targeting B lymphoma cells with doxorubicin-loaded liposomal nanoparticles displaying high-affinity glycan ligands of CD22. The targeted liposomes are actively bound and endocytosed by CD22 on B cells, and significantly extend life in a xenograft model of human B-cell lymphoma. Moreover, they bind and kill malignant B cells from peripheral blood samples obtained from patients with hairy cell leukemia, marginal zone lymphoma, and chronic lymphocytic leukemia. The results demonstrate the potential for using a carbohydrate recognition-based approach for efficiently targeting B cells in vivo that can offer improved treatment options for patients with B-cell malignancies.

2-Chlorodeoxyadenosine activity in patients with untreated chronic lymphocytic leukemia.

PURPOSE 2-chlorodeoxyadenosine (2-CdA; cladribine) is a purine analog with activity in patients with chronic lymphocytic leukemia (CLL) who fail to respond to alkylator therapy. We conducted a phase II trial of 2-CdA in previously untreated CLL patients. PATIENTS AND METHODS 2-CdA was administered to 20 patients with previously untreated CLL as a 0.1-mg/kg/d 7-day continuous intravenous infusion every 28 to 35 days until maximum response or prohibitive toxicity. RESULTS A median of four courses (range, one to nine) was administered to each patient. Five patients (25%) achieved a complete response and 12 (60%) achieved a partial response, for an overall response rate of 85%. The median response follow-up duration was 8+ months (range, 3 to 27). Myelosuppression was the principal toxicity. Four of 20 patients (20%) experienced grade III or IV thrombocytopenia. Three patients, all of whom received corticosteroid therapy, developed opportunistic infections at a median of 19 months following discontinuation of 2-CdA therapy. CONCLUSION 2-CdA has major activity in patients with previously untreated CLL, and the lower response rates seen in previously treated patients may be due in part to poor marrow reserve from prior therapy. Determination of the relative effectiveness of 2-CdA, fludarabine, and chlorambucil in the treatment of CLL patients will require a randomized trial.

2-Chlorodeoxyadenosine Treatment of Refractory Chronic Lymphocytic Leukemia.

Ninety patients with advanced refractory chronic lymphocytic leukemia (CLL) were treated with 2-chlorodeoxyadenosine (2-CdA) administered either as a 0.1 mg/kg/day 7-day continuous intravenous infusion or as a 0.028 mg to 0.14 mg/kg/day 2-hour bolus for 5 consecutive days. One patient had stage A disease, seven patients had stage B disease, and 82 patients had stage C disease. Twenty-seven patients were female and 63 were male, with an age range of 40 to 84 years, median 63 years. All patients had received prior therapy and failed, with a range of one to four and a median of two prior therapies. Six patients had previously failed fludarabine therapy. Four patients (4%) experienced complete remissions, and 40 patients (40%) experienced partial remissions, yielding an over all response rate of 44%. The median duration of response was four months, with a range of two to 30 months. Of 50 patients who were non-responders, 27 (54%) had a greater than 50% sustained reduction in the absolute lymphocyte count despite insufficient improvement in hemoglobin concentration or platelet count to achieve a response status. Therapy was well tolerated with myelosuppression being the principal toxicity. Twenty-two patients (24%) experienced thrombocytopenia and 16 patients (18%) had documented infections. We confirm our early pilot results with 2-CdA demonstrating in a large group of patients that 2-CdA achieves a significant response rate with two different drug administration schemes in failed CLL patients. Responses are achieved with acceptable toxicity. 2-CdA merits further evaluation in previously untreated patients and in combination regimens for failed patients.

2-Chlorodeoxyadenosine: A Newer Purine Analog Active in the Treatment of Indolent Lymphoid Malignancies

2-Chlorodeoxyadenosine ([2-CdA], cladribine; Leustatin, Ortho Biotech, Raritan, New Jersey), along with fludarabine [1] and 2-deoxycoformycin [2], is a newer purine analog with major activity in the treatment of indolent lymphoid malignancies. Fludarabine (Fludara, Berlex Laboratories, Alameda, California) is approved by the Food and Drug Administration for the treatment of patients with chronic lymphocytic leukemia who are refractory to alkylating agents [3], 2-deoxycoformycin (Nipent, Parke Davis; Morris Plains, New Jersey) for the treatment of interferon-refractory hairy cell leukemia, and 2-CdA for patients with untreated or interferon-refractory hairy cell leukemia. The development of 2-CdA emerged from an improved understanding of the mechanisms of lymphopenia in adenosine deaminase-deficient children with severe combined immunodeficiency disease. We review the development, structure, mechanism of action, pharmacologic features, and the clinical trial results of this important new chemotherapeutic agent. Lessons from Adenosine Deaminase Deficiency In 1972, Giblett and colleagues [4] made the serendipitous but seminal observation that some infants with severe combined immunodeficiency disease were adenosine deaminase deficient. Cohen and colleagues [5] later established the relation between the intracellular accumulation of deoxyribonucleotides (resulting from adenosine deaminase deficiency) and lymphocytotoxicity. Carson and colleagues [6] evaluated a panel of purine deoxynucleosides synthesized to be resistant to deamination by adenosine deaminase for toxicity in vitro and identified 2-CdA as the most potent. 2-Chlorodeoxyadenosine induces a lymphopenic state, similar to that seen in adenosine deaminase deficiency, by resisting deamination and thereby accumulating in its triphosphate form with resultant lymphocytotoxicity. Structure and Synthesis 2-Chlorodeoxyadenosine is a deoxyadenosine analog, consisting of substitution of a chlorine atom for the hydrogen atom at the 2-position of the purine ring (Figure 1). It was first synthesized by Christensen and colleagues [7], using direct fusion alkylation of 2,6-dichloropurine. Later, Carson and colleagues [6] synthesized 2-CdA from 2-chloroadenine and thymidine using a transdeoxyribosylase from Lactobacillus helveticus. The drug in commercial use today is synthesized nonenzymatically using a sodium salt glycosylation procedure. Figure 1. Molecular structures of deoxyadenosine and 2-chlorodeoxyadenosine. Mechanisms of Action Adenosine deaminase catalyzes the irreversible deamination of deoxyadenosine to deoxyinosine (Figure 2). Deoxycytidine kinase phosphorylates purine deoxyribonucleotides, whereas 5-nucleotidase dephosphorylates them. Lymphocytes have high deoxycytidine kinase to 5-nucleotidase ratios, favoring triphosphate formation [8] and making them ideal target cells for 2-CdA. Figure 2. Deoxyadenosine metabolism. d. adenosine DCK NT d. AMP d. ADP d. ATP ADA d. adenosine d. inosine PNP XO 2-Chlorodeoxyadenosine enters the cell through an efficient transport system and is phosphorylated by deoxycytidine kinase. 2-Chlorodeoxyadenosine triphosphate is a potent inhibitor of ribonucleotide reductase and DNA polymerase- (Figure 3). 2-Chlorodeoxyadenosine triphosphate accumulation also depletes the intracellular pool of deoxynucleotides [9]. In actively dividing cells, DNA synthesis is then impaired by the preferential use of 2-chlorodeoxyadenosine triphosphate by DNA polymerase and the retardation of DNA chain elongation [10]. Figure 3. 2-Chlorodeoxyadenosine mechanism of action. 2-CdA ADA DCK 5-NT 2-CdAMP 2-CdADP 2-CdATP RNR NAD ATP 2-Chlorodeoxyadenosine is unique compared with traditional antimetabolites in being equally active against both dividing and resting cells [11, 12]. A different mechanism of action must therefore operate in resting cells because ribonucleotide reductase is only expressed at low levels. The DNA strand breaks that gradually accumulate with time activate two enzyme systems: poly-(ADP-ribose) polymerase-consuming nicotinamide adenine dinucleotide and adenosine triphosphate [12, 13] and a Ca++/Mg ++-dependent endonuclease that produces double-stranded DNA breaks at internucleosomal regions [14]. The cleavage of DNA into oligonucleosomal fragments follows, which is the hallmark of apoptosis, a form of programmed cell death [15-17]. Apoptosis Distinct from necrosis, apoptosis is a physiologic mechanism of cell death [18]. It is the mechanism by which senescent or abnormal cells that could interfere with organ function or develop into cancer are removed. Morphologically, apoptosis is characterized by compaction of chromatin against the nuclear membrane, cell shrinkage, and nuclear and cytoplasmic budding to form membrane-bound fragments, called apoptosis bodies, which are phagocytosed by adjacent cells or macrophages [17]. This process is completed in the absence of inflammatory changes. The tendency of a cancer cell to undergo apoptosis may determine the sensitivity of tumors with low growth fractions to chemotherapy. Indolent lymphoid tumors have high expression of the bcl-2 oncogene, known to enhance cell survival through its interference with apoptosis [19]. The induction of apoptosis by 2-CdA stimulates even greater interest in this agent for potential use in these bcl-2 expressing lymphoid malignancies. A possible relation between the expression of bcl-2 and susceptibility of the malignant cell to 2-CdA is under study. Preclinical Studies and Pharmacologic Features Preclinical studies showed that both B- and T-lymphoblastoid cell lines were sensitive to 2-CdA but that T-lymphoblastoid cell lines were more vulnerable [6]. Clinically, this difference is not apparent, and, in fact, B- lymphocyte-derived disorders are highly responsive. Prolonged exposure of resting peripheral blood lymphocytes to 2-CdA in vitro resulted in greater lymphocytotoxicity than did brief incubations [20], which led to the selection of a continuous intravenous infusion schedule for the initial clinical trials. The single 50% lethal dose of 2-CdA given intraperitoneally to mice was 150 mg/kg body weight and with daily administration for 5 days, the 50% lethal dose was 100 mg/kg [21]. 2-Chlorodeoxyadenosine prolonged the life of mice with L1210 leukemia [6]. Doses of 1 mg/kg given by continuous intravenous infusion to monkeys for 7 days caused severe diarrhea and granulocytopenia. 2-Chlorodeoxyadenosine is cleared by mammalian kidneys, probably secreted through the renal organic cation carrier system, and its elimination is according to a two-compartment model, with and -half-lives of 35 minutes and 6.7 hours, respectively [22]. Plasma 2-CdA concentrations of 20 to 30 nmol in patients with lymphoid malignancies were achieved with the standard infusion dose of 0.1 mg/kg per day by continuous infusion, which exceeds the 50% inhibition of growth for some human malignant lymphoblast cell lines incubated with 2-CdA in vitro [20]. A bolus method of 2-CdA administration was developed based on pharmacokinetic studies showing high concentrations and prolonged intracellular retention of 2-chlorodeoxyribonucleotides in chronic lymphocytic leukemia [23]. This method of drug delivery was devised to facilitate the outpatient administration of 2-CdA and to avoid the need for central catheters and infusion devices [24]. The bioavailability of 2-CdA given subcutaneously is 100% and, when administered orally, is 50%, although there are considerable differences among patients. Oral absorption is not enhanced by suppression of gastric acid [25]. 2-Chlorodeoxyadenosine penetrates the cerebrospinal fluid [26] with levels that are 25% of the plasma levels [27]. Clinical StudiesPhase 1 2-Chlorodeoxyadenosine dose-escalation studies showed myelosuppression, with considerable hematopoietic stem cell toxicity to be dose limiting. A maximum tolerated dose of 0.1 mg/kg per day for 7 days by continuous infusion (using the conversion factor of 40, 0.1 mg/kg is equivalent to 4 mg/m2 [28]) was established and was associated with a 25% incidence of myelosuppression [20]. Single courses of the drug at this dose caused transient marrow suppression with granulocytopenia and thrombocytopenia, especially in patients who were pancytopenic before the start of 2-CdA therapy. When repeated courses of 2-CdA were administered, cumulative thrombocytopenia became the limiting toxicity in 20% to 30% of patients, which persisted in some patients for more than 6 months. At this dose, no nausea, vomiting, alopecia, nephrotoxicity, hepatotoxicity, pulmonary and cardiac toxicity, or neurotoxicity was observed. Severe and sometimes irreversible nephrotoxicity and neurotoxicity with paresis were encountered when 2-CdA was administered at 0.4 to 0.5 mg/kg (16 to 20 mg/m2) per day for 7 to 14 days by continuous infusion in combination with high-dose cyclophosphamide and total body irradiation in preparation for allogeneic bone marrow transplantation. The contribution of 2-CdA compared with total body irradiation or high-dose cyclophosphamide in conjunction with the 2-CdA to these toxicities is unclear. In the initial studies, the actual dose of 2-CdA administered to patients was 0.09 mg/kg per day because doses were standardized using the extinction coefficient of chloroadenine, which proved to be slightly lower than that of 2-CdA [29]. Subsequently, all patients received 0.1 mg/kg per day because of synthesis and formulation changes, representing most patients treated in the clinical development of this drug. The maximum tolerated dose for 2-CdA delivered as a 7-day intravenous infusion to patients with nonhematologic malignancies was also 0.1 mg/kg per day. Neurologic events occurred in two patients, both with malignant melanoma, one treated with 0.15 mg/kg per day of 2-CdA and the other with 0.2 mg/kg per day. A direct neurotoxic role for 2-CdA was not absolutely established because of other associated conditio

Targeting Mutant BRAF in Relapsed or Refractory Hairy-Cell Leukemia

BACKGROUND BRAF V600E is the genetic lesion underlying hairy-cell leukemia. We assessed the safety and activity of the oral BRAF inhibitor vemurafenib in patients with hairy-cell leukemia that had relapsed after treatment with a purine analogue or who had disease that was refractory to purine analogues. METHODS We conducted two phase 2, single-group, multicenter studies of vemurafenib (at a dose of 960 mg twice daily)--one in Italy and one in the United States. The therapy was administered for a median of 16 weeks in the Italian study and 18 weeks in the U.S. study. Primary end points were the complete response rate (in the Italian trial) and the overall response rate (in the U.S. trial). Enrollment was completed (28 patients) in the Italian trial in April 2013 and is still open (26 of 36 planned patients) in the U.S. trial. RESULTS The overall response rates were 96% (25 of 26 patients who could be evaluated) after a median of 8 weeks in the Italian study and 100% (24 of 24) after a median of 12 weeks in the U.S. study. The rates of complete response were 35% (9 of 26 patients) and 42% (10 of 24) in the two trials, respectively. In the Italian trial, after a median follow-up of 23 months, the median relapse-free survival was 19 months among patients with a complete response and 6 months among those with a partial response; the median treatment-free survival was 25 months and 18 months, respectively. In the U.S. trial, at 1 year, the progression-free survival rate was 73% and the overall survival rate was 91%. Drug-related adverse events were usually of grade 1 or 2, and the events most frequently leading to dose reductions were rash and arthralgia or arthritis. Secondary cutaneous tumors (treated with simple excision) developed in 7 of 50 patients. The frequent persistence of phosphorylated ERK-positive leukemic cells in bone marrow at the end of treatment suggests bypass reactivation of MEK and ERK as a resistance mechanism. CONCLUSIONS A short oral course of vemurafenib was highly effective in patients with relapsed or refractory hairy-cell leukemia. (Funded by the Associazione Italiana per la Ricerca sul Cancro and others; EudraCT number, 2011-005487-13; ClinicalTrials.gov number NCT01711632.).

Bolus administration of cladribine in the treatment of Waldenström macroglobulinaemia

This phase II clinical trial evaluated bolus cladribine as a single agent in Waldenström macroglobulinaemia (WM).