Walter N. Hittelman

THE EFFECT OF CELECOXIB, A CYCLOOXYGENASE-2 INHIBITOR, IN FAMILIAL ADENOMATOUS POLYPOSIS

BACKGROUND Patients with familial adenomatous polyposis have a nearly 100 percent risk of colorectal cancer. In this disease, the chemopreventive effects of nonsteroidal antiinflammatory drugs may be related to their inhibition of cyclooxygenase-2. METHODS We studied the effect of celecoxib, a selective cyclooxygenase-2 inhibitor, on colorectal polyps in patients with familial adenomatous polyposis. In a double-blind, placebo-controlled study, we randomly assigned 77 patients to treatment with celecoxib (100 or 400 mg twice daily) or placebo for six months. Patients underwent endoscopy at the beginning and end of the study. We determined the number and size of polyps from photographs and videotapes; the response to treatment was expressed as the mean percent change from base line. RESULTS At base line, the mean (+/-SD) number of polyps in focal areas where polyps were counted was 15.5+/-13.4 in the 15 patients assigned to placebo, 11.5+/-8.5 in the 32 patients assigned to 100 mg of celecoxib twice a day, and 12.3+/-8.2 in the 30 patients assigned to 400 mg of celecoxib twice a day (P=0.66 for the comparison among groups). After six months, the patients receiving 400 mg of celecoxib twice a day had a 28.0 percent reduction in the mean number of colorectal polyps (P=0.003 for the comparison with placebo) and a 30.7 percent reduction in the polyp burden (the sum of polyp diameters) (P=0.001), as compared with reductions of 4.5 and 4.9 percent, respectively, in the placebo group. The improvement in the extent of colorectal polyposis in the group receiving 400 mg twice a day was confirmed by a panel of endoscopists who reviewed the videotapes. The reductions in the group receiving 100 mg of celecoxib twice a day were 11.9 percent (P=0.33 for the comparison with placebo) and 14.6 percent (P=0.09), respectively. The incidence of adverse events was similar among the groups. CONCLUSIONS In patients with familial adenomatous polyposis, six months of twice-daily treatment with 400 mg of celecoxib, a cyclooxygenase-2 inhibitor, leads to a significant reduction in the number of colorectal polyps.

Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-Trans-retinoic acid)

BACKGROUND AND METHODS Patients with acute promyelocytic leukemia have a characteristic (15;17) translocation, with a breakpoint on chromosome 17 in the region of the retinoic acid receptor-alpha (RAR-alpha). Since this receptor has been shown to be involved with growth and differentiation of myeloid cells in vitro, and since recent clinical studies have reported that tretinoin (all-trans-retinoic acid) induces complete remission in patients with acute promyelocytic leukemia we studied the effects of tretinoin on cellular maturation and molecular abnormalities in patients undergoing the induction of remission with this agent. RESULTS Eleven patients with acute promyelocytic leukemia were treated with tretinoin administered orally at a dose of 45 mg per square meter of body-surface area per day. Nine of the 11 patients entered complete remission. In two patients, complete remission was preceded by striking leukocytosis that then resolved despite continued drug treatment. Serial studies of cellular morphologic features, cell-surface immunophenotypic analysis, and fluorescence in situ hybridization with a chromosome 17 probe revealed that clinical response was associated with maturation of the leukemic clone. All patients who responded to treatment who were tested by Northern blot analysis had expression of aberrant RAR-alpha. As patients entered complete remission, the expression of the abnormal RAR-alpha message decreased markedly; however, it was still detectable in several patients after complete morphologic and cytogenetic remission had been achieved. CONCLUSIONS Tretinoin is a safe and highly effective agent for inducing complete remission in patients with acute promyelocytic leukemia. Clinical response to this agent is associated with leukemic-cell differentiation and is linked to the expression of an aberrant RAR-alpha nuclear receptor. Molecular detection of the aberrant receptor may serve as a useful marker for residual leukemia in patients with this disease.

A missense mutation in KIT kinase domain 1 correlates with imatinib resistance in gastrointestinal stromal tumors

KIT gain of function mutations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of ABL, platelet-derived growth factor receptor (PDGFR), and KIT and represents a new paradigm of targeted therapy against GISTs. Here we report for the first time that, after imatinib treatment, an additional specific and novel KIT mutation occurs in GISTs as they develop resistance to the drug. We studied 12 GIST patients with initial near-complete response to imatinib. Seven harbored mutations in KIT exon 11, and 5 harbored mutations in exon 9. Within 31 months, six imatinib-resistant rapidly progressive peritoneal implants (metastatic foci) developed in five patients. Quiescent residual GISTs persisted in seven patients. All six rapidly progressive imatinib-resistant implants from five patients show an identical novel KIT missense mutation, 1982T→C, that resulted in Val654Ala in KIT tyrosine kinase domain 1. This novel mutation has never been reported before, is not present in pre-imatinib or post-imatinib residual quiescent GISTs, and is strongly correlated with imatinib resistance. Allelic-specific sequencing data show that this new mutation occurs in the allele that harbors original activation mutation of KIT.

Stimulation of Megakaryocyte and Platelet Production by a Single Dose of Recombinant Human Thrombopoietin in Patients with Cancer

Thrombocytopenia is an important clinical problem in the management of patients in hematology and oncology practices. In the United States, the use of platelet transfusions to manage severe thrombocytopenia has steadily increased: Approximately 4 million units were transfused in 1982, and more than 8 million units were transfused in 1992 [1, 2]. This marked increase in the need for platelets has paralleled advances in organ transplantation, bone marrow transplantation, cardiac surgery, and the use of dose-intensive therapy in the treatment of chemosensitive malignant conditions. Although platelet transfusions may decrease the risk for fatal bleeding complications, repeated transfusions increase the risk for transmission of bacterial and viral pathogens, transfusion reactions, and transfusion-associated graft-versus-host disease. These transfusions also contribute to increasing health care costs and inconvenience to patients [3]. Thus, an agent that can increase platelet production and prevent or attenuate thrombocytopenia would be an important advance. Thrombopoietin, the ligand for the c-Mpl receptor (found on platelets and megakaryocyte progenitors), was recently cloned by several investigators and was shown to be a primary regulator of platelet production in vivo [4-8]. Thrombopoietin promotes both the proliferation of megakaryocyte progenitors and their maturation into platelet-producing megakaryocytes. In preclinical studies done in normal mice and nonhuman primates, thrombopoietin increased platelet counts to a level higher than those previously achieved with other thrombopoietic cytokines [9, 10]. Moreover, in a murine model for myelosuppression, recombinant thrombopoietin given as a single dose decreased the nadir and accelerated platelet recovery in mice that had been rendered pancytopenic by sublethal radiation and chemotherapy [11]. In these studies, more prolonged treatment (for as long as 8 days) provided no additional benefit and was associated with marked thrombocytosis during the recovery phase. On the basis of these observations, we initiated a phase I and II clinical and laboratory investigation of recombinant human thrombopoietin in patients with cancer who were at high risk for severe chemotherapy-induced thrombocytopenia. This trial was divided into two parts: Part I studied thrombopoietin given before chemotherapy, and part II studied thrombopoietin given after chemotherapy. The objective of part I, the results of which are reported here, was to assess the hematopoietic effects, pharmacodynamics, and clinical tolerance of this novel agent in patients who had normal hematopoietic function before chemotherapy. Methods Patients Patients with sarcoma who had never had chemotherapy, were suitable candidates for subsequent chemotherapy, and did not have rapidly progressive disease were eligible for this trial. Patients were required to have a Karnofsky performance status score of 80 or more, adequate bone marrow (absolute neutrophil count 1.5 109/L; platelet count 150 109/L and 450 109/L), adequate renal function (serum creatinine level 120 mol/L), and adequate hepatic function (alanine aminotransferase level < 3 times normal; bilirubin level < 1.5 times normal). Patients with a history of thromboembolic or bleeding disorders, significant cardiac disease, or previous pelvic radiation were excluded. Written informed consent was obtained from all patients before study entry in accordance with institutional guidelines. Design During the phase I dose-ranging portion of this clinical cohort study, thrombopoietin was administered as a single intravenous dose 3 weeks before chemotherapy. At study entry, three patients were assigned to each of four dose levels (0.3, 0.6, 1.2, and 2.4 g/kg of body weight). Patients who had no dose-limiting toxicity and did not develop neutralizing antibodies to thrombopoietin were eligible to receive thrombopoietin at the same doses after chemotherapy. Recombinant Human Thrombopoietin The thrombopoietin used in this study was provided by Genentech, Inc. (South San Francisco, California). Thrombopoietin is a full-length glycosylated molecule produced in a genetically modified mammalian cell line and purified by standard techniques. It was mixed with preservative-free normal saline as a diluent for injections. Clinical and Laboratory Monitoring Before and during the clinical trial, patients were monitored by complete histories; physical examinations; and laboratory tests, including a complete blood cell count with differential counts, serum chemistry, coagulation profile, urinalysis, assessment of thrombopoietin antibody formation, chest radiography, and electrocardiography. Blood counts were obtained daily for the first 5 days and then at least three times per week. Peripheral smears were examined serially for platelet morphology. Platelet counts and the average size of platelets (mean platelet volume) were derived from 64-channel platelet histograms. Bone marrow aspiration and biopsy were done before and 1 week after thrombopoietin treatment. The bone marrow specimens were initially fixed in 10% neutral formalin, embedded in paraffin, cut into sections 5 m thick, and stained with hematoxylin-eosin for morphologic analysis and with Masson trichrome for analysis of collagen fiber content. Fresh, air-dried smears of bone marrow were stained with Wright-Giemsa. Bone marrow samples were examined for overall cellularity and morphology in a blinded manner. Megakaryocyte counts were measured by choosing 10 high-power (40x) fields in areas without artifactual zones or trabecula. The relative size of the megakaryocyte was assessed by examining bone marrow aspirate smears using the Magiscan Image Analysis System (Compix, Cranberry, Pennsylvania). Bone marrow aspirates were also assayed for hematopoietic progenitor cell number and cycle status, for content of CD34+ and CD41+ cell subsets (by flow cytometry), and for megakaryocyte ploidy (by flow cytometry). Blood samples were assayed for hematopoietic progenitor cell number and for platelet function. Pharmacokinetics Profiles Serum samples were collected before and at 2, 5, 10, 60, and 90 minutes and 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 hours after thrombopoietin administration. Concentration-time profile at each dose level was evaluated by using standard pharmacokinetics methods. Serum thrombopoietin levels were quantitated by enzyme-linked immunosorbent assay for thrombopoietin [12]. Hematopoietic Progenitor Cell Assays Assays for colony-forming unit-granulocyte-macrophage (CFU-GM); burst-forming unit-erythroid (BFU-E); and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) using low-density bone marrow [13] and peripheral blood cells [14] were done with methyl cellulose assays. The percentage of bone marrow CFU-GM and BFU-E in DNA synthesis (S-phase of cell cycle) was measured by a high-specific-activity tritiated thymidine suicide technique [15]. Assays for colony-forming unit-megakaryocyte (CFU-MK) and burst-forming unit-megakaryocyte (BFU-MK) were done using a fibrin clot assay [16]. Ploidy Analysis Megakaryocyte-enriched cell fractions were prepared from bone marrow cell suspensions by using a Percoll gradient technique. Ploidy was determined by flow cytometric measurement of the relative DNA content after staining with propidium iodide in hypotonic citrate solution [17]. Cells were also stained with anti-CD41b (8D9)-FITC (SyStemix, Palo Alto, California) to allow gating on CD41b+ megakaryocytes. At least 3000 CD41+ events were collected for each sample. The percentage of CD41+ cells in ploidy class was determined from the fluorescence-activated cell-sorting dot plots. Platelet Function Platelet aggregation was measured in response to three agonists: adenosine diphosphate (final concentration, 20 g/mL), collagen (6 g/mL), and thrombin (5 g/mL). Standard methods were used [18]. The concentrations of agonists were chosen on the basis of previous in vitro studies done on blood from normal controls. The instruments used for the assays were the Bio/Data Pap 4A (Horsham, Pennsylvania) and the Crono-log 560CA (Havertown, Pennsylvania). Immunophenotypic Analysis Immunophenotypic analysis was done using anti-CD34 (Becton Dickinson, San Jose, California) and anti-CD41 monoclonal antibodies (Immunotech, Westbrook, Maine) by a standard dual-color flow cytometry technique [19]. Statistical Analysis Continuous variables were compared by using the Wilcoxon matched-pairs signed-rank test. Trends for possible dose-response relation were evaluated using the Spearman rank correlation coefficients (rS) between dose and outcome. Industry Role Thrombopoietin and partial funding for the study were provided by Genentech, Inc. The study was a collaborative effort between the principal investigator and the industrial sponsor. Data collection, data analysis, the writing of the manuscript, and the decision to publish the manuscript were under the control of the principal investigator. The manuscript was reviewed by the industrial sponsor before submission. Results Twelve chemotherapy-naive patients (7 men and 5 women) with sarcoma of diverse histologic sub-types were entered into the dose-ranging portion of this phase I trial, which studied thrombopoietin before chemotherapy. All patients were considered evaluable for clinical tolerance and response to thrombopoietin. The median age of these patients was 42 years (range, 16 to 63 years), and the median Karnofsky performance status score was 90 (range, 80 to 100). Four patients had previously received radiation therapy, and eight had previously had surgery. Peripheral Blood Counts Treatment with a single dose of thrombopoietin was associated with increases (1.3-fold to 3.6-fold) in platelet counts (baseline mean, 264 109/L; maximal mean, 592 109/L) (P = 0.002). The increase in platelet count was seen at all dose levels (Figure 1) in all patients. The peak response i

Expression of Blood-Group Antigen A — A Favorable Prognostic Factor in Non-Small-Cell Lung Cancer

BACKGROUND New prognostic factors are needed to guide the treatment of patients with non-small-cell lung cancer. We evaluated the prognostic value of altered expression of ABH blood-group antigens, which has been implicated in the multistep process of carcinogenesis and tumor progression. METHODS The presence of blood-group antigens was assessed immunohistochemically in paraffin-embedded tumor samples from 164 patients who underwent curative surgery for non-small-cell lung cancer from 1980 through 1982. Monoclonal antibodies were used to detect the A and B antigens, and Ulex europaeus agglutinin I to detect H antigen. RESULTS Survival of the 28 patients with blood type A or AB who had primary tumors negative for blood-group antigen A was significantly shorter than that of the 43 patients with antigen A-positive tumors (P less than 0.001) and of the 93 patients with blood type B or O (P = 0.002). The respective median survival times were 15, 71, and 39 months. Disease progressed significantly earlier in the 28 patients with tumors negative for blood-group antigen A than in the antigen A-positive patients (P less than 0.001). Expression of blood-group antigen B or H in tumor cells did not correlate with survival. Cox proportional-hazards regression analysis showed that expression of blood-group antigen A in tumor cells added significantly to the prediction of overall survival provided by other known prognostic factors among the patients with blood type A or AB (P = 0.004). CONCLUSIONS Expression of blood-group antigen A in tumor cells is an important favorable prognostic factor in patients with non-small-cell lung cancer. This variable needs to be considered in the design of future trials of therapy.

ErbB2 Increases Vascular Endothelial Growth Factor Protein Synthesis via Activation of Mammalian Target of Rapamycin/p70S6K Leading to Increased Angiogenesis and Spontaneous Metastasis of Human Breast Cancer Cells

ErbB2 overexpression in breast tumors results in increased metastasis and angiogenesis and reduced survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, we did a spontaneous metastasis assay using MDA-MB-435 human breast cancer cells stably transfected with constitutively active ErbB2 kinase (V659E), a kinase-dead mutant of ErbB2 (K753M), or vector control (neo). Mice injected with V659E had increased metastasis incidence and tumor microvessel density than mice injected with K753M or control. Increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro. V659E produced increased vascular endothelial growth factor (VEGF) through increased VEGF protein synthesis. This was mediated through signaling events involving extracellular signal-regulated kinase, phosphatidylinositol 3-kinase/Akt, mammalian target of rapamycin (mTOR), and p70S6K. The V659E xenografts also had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with controls. To validate the clinical relevance of these findings, we examined 155 human breast tumor samples. Human tumors that overexpressed ErbB2, which have been previously shown to have higher VEGF expression, showed significantly higher p70S6K phosphorylation as well. Increased VEGF expression also significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Our findings show that ErbB2 increases VEGF protein production by activating p70S6K in cell lines, xenografts, and in human cancers and suggest that these signaling molecules may serve as targets for antiangiogenic and antimetastatic therapies.

Genetic Variations in Radiation and Chemotherapy Drug Action Pathways Predict Clinical Outcomes in Esophageal Cancer

PURPOSE Understanding how specific genetic variants modify drug action pathways may provide informative blueprints for individualized chemotherapy. METHODS We applied a pathway-based approach to examine the impact of a comprehensive panel of genetic polymorphisms on clinical outcomes in 210 esophageal cancer patients. RESULTS In the Cox proportional hazards model, MTHFR Glu429Ala variant genotypes were associated with significantly improved survival (hazard ratio [HR] = 0.56; 95% CI, 0.35 to 0.89) in patients treated with fluorouracil (FU). The 3-year survival rates for patients with the variant genotypes and the wild genotypes were 65.26% and 46.43%, respectively. Joint analysis of five polymorphisms in three FU pathway genes showed a significant trend for reduced recurrence risk and longer recurrence-free survival as the number of adverse alleles decreased (P = .004). For patients receiving platinum drugs, the MDR1 C3435T variant allele was associated with significantly reduced recurrence risk (HR = 0.25; 95% CI, 0.10 to 0.64) and improved survival (HR = 0.44; 95% CI, 0.23 to 0.85). In nucleotide excision repair genes, there was a significant trend for a decreasing risk of death with a decreasing number of high-risk alleles (P for trend = .0008). In base excision repair genes, the variant alleles of XRCC1 Arg399Gln were significantly associated with the absence of pathologic complete response (odds ratio = 2.75; 95% CI, 1.14 to 6.12) and poor survival (HR = 1.92; 95% CI, 1.00 to 3.72). CONCLUSION Several biologically plausible associations between individual single nucleotide polymorphisms and clinical outcomes were found. Our data also strongly suggest that combined pathway-based analysis may provide valuable prognostic markers of clinical outcomes.

The Repair of Double-Strand DNA Breaks Correlates with Radiosensitivity of L5178Y-S and L5178Y-R Cells

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.

Stimulation of Myelopoiesis in Patients with Aplastic Anemia by Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor

Aplastic anemia is a syndrome in which pancytopenia occurs in the presence of hypocellularity of the bone marrow. To assess the biologic activities of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) in aplastic anemia, we gave GM-CSF (60 to 500 micrograms per square meter of body-surface area) to 10 patients with moderate or severe disease, by continuous intravenous infusion daily for two weeks, and repeated the treatment after a two-week rest period. The treatment increased the white-cell count (1.6- to 10-fold) in all patients, primarily because of an increase in the numbers of neutrophils (1.5 to 20-fold), eosinophils (12- to greater than 70-fold), and monocytes (2- to 32-fold). Rates of hydrogen peroxide production in purified granulocyte fractions increased during GM-CSF treatment. Increases in bone marrow cellularity, myeloid precursor cells, and myeloid:erythroid cell ratios accompanied the white-cell response. Despite the in vivo response of the white-cells, the concentration of colony-forming cells remained the same. Measurable concentrations of interleukin-2 (2 to 15 units per milliliter) were found in the serum of 8 patients, and high levels of erythropoietin (81 to 1200 IU per liter) were found in 10 patients. The predominant side effects were constitutional symptoms. These results indicate that recombinant human GM-CSF is effective in stimulating myelopoiesis in patients with severe aplastic anemia and may benefit some patients in whom the disorder is refractory to standard forms of therapy.

Frequent inactivation of p16(INK4α) in oral premalignant lesions

Head and neck carcinogenesis is believed to be a multistep process, whereby genetic events accumulate in the carcinogen-exposed field at risk, resulting in distinct phenotypic premalignant changes that eventually evolve into invasive cancer. Frequent loss of heterozygosity (LOH) at the chromosome 9p21 region and inactivation of p16INK4a by different mechanisms have been described in head and neck squamous cell carcinoma (HNSCC). Recently, we reported that loss of 9p21 is also frequent in oral premalignant lesions. To investigate potential inactivation of p16INK4a in these premalignant lesions, we analysed 74 biopsies from 36 patients by immunohistochemistry (IHC) for expression of the p16 protein. Loss of p16 expression was found in 28 (38%) of the lesion biopsies from 17 patients (47%). LOH at the D9s171, a marker in the 9p21 region, was observed in 19 lesion biopsies from 12 cases and correlated with absence of p16 by IHC in 11 (92%) of the 12 comparable cases and 15 (79%) of 19 lesion biopsies. By direct sequencing of ten lesion biopsies from ten individuals with LOH at D9s171 for p16INK4a exon 2, one non-sense mutation at codon 88 (GGA→TGA) was identified. Our data suggest that inactivation of p16INK4a may play an important role in early head and neck cancer development.