|
Advertisement |
|
|
||
 |
|
|||||
|
|||||
|
 |
||||||
|
||||||
|
||||||
|
Journal of Clinical Oncology, Vol 24, No 15 (May 20), 2006: pp. 2283-2289 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.04.5716 Enterocolitis in Patients With Cancer After Antibody Blockade of Cytotoxic T-Lymphocyte–Associated Antigen 4
From the Surgery Branch and Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD; and Medarex Inc, Princeton NJ Address reprint requests to James C. Yang, MD, Surgery Branch, National Cancer Institute, National Institutes of Health, CRC, Room 3W, 10 Center Dr, Bethesda, MD 20892-1201; e-mail: James_Yang{at}nih.gov
PURPOSE: Cytotoxic T-lymphocyte–associated antigen 4 (CTLA4) is an inhibitory receptor on T cells. Knocking out CTLA4 in mice causes lethal lymphoproliferation, and polymorphisms in human CTLA4 are associated with autoimmune disease. Trials of the anti-CTLA4 antibody ipilimumab (MDX-010) have resulted in durable cancer regression and immune-mediated toxicities. A report on the diagnosis, pathology, treatment, clinical outcome, and significance of the immune-mediated enterocolitis seen with ipilimumab is presented. PATIENTS AND METHODS: We treated 198 patients with metastatic melanoma (MM) or renal cell carcinoma (RCC) with ipilimumab. RESULTS: The overall objective tumor response rate was 14%. We observed several immune mediated toxicities including dermatitis, enterocolitis, hypophysitis, uveitis, hepatitis, and nephritis. Enterocolitis, defined by grade 3/4 clinical presentation and/or biopsy documentation, was the most common major toxicity (21% of patients). It presented with diarrhea, and biopsies showed both neutrophilic and lymphocytic inflammation. Most patients who developed enterocolitis responded to high-dose systemic corticosteroids. There was no evidence that steroid administration affected tumor responses. Five patients developed perforation or required colectomy. Four other patients with steroid-refractory enterocolitis appeared to respond promptly to tumor necrosis factor alpha blockade with infliximab. Objective tumor response rates in patients with enterocolitis were 36% for MM and 35% for RCC, compared with 11% and 2% in patients without enterocolitis, respectively (P = .0065 for MM and P = .0016 for RCC). CONCLUSION: CTLA4 seems to be a significant component of tolerance to tumor and in protection against immune mediated enterocolitis and these phenomena are significantly associated in cancer patients.
Cytotoxic T-lymphocyte–associated antigen 4 (CTLA4) is a cell surface receptor initially cloned from a cDNA library from a murine cytotoxic T-lymphocyte.1 Its ligands are CD80 and CD86 which also participate in lower affinity interactions with the costimulatory T-cell receptor CD28. Rather than costimulate, CTLA4 functions as an inducible receptor with T-cell inhibitory activity.2-5 Thus its primary role is to down-regulate T-cell activation. CTLA4 was also found constitutively expressed on inhibitory CD25+CD4+ regulatory T cells (Treg) and CTLA4 signaling was necessary in Treg control of intestinal inflamation.6 Targeted destruction of the CTLA4 gene in mice causes lymphoproliferation and autoimmune disease and antimurine CTLA4 antibodies induced antitumor activity, particularly when combined with antitumor vaccination.5,7,8 This led to clinical trials of a fully human immunoglobulin G1 antibody against CTLA4, ipilimumab (formerly MDX-010; Medarex Inc, Princeton, NJ). In patients with melanoma or ovarian cancer who also had antitumor vaccination, tumor necrosis and cellular infiltration was reported after ipilimumab administration,9 and other studies have also documented durable tumor regression by standard criteria. Phan et al reported 14 patients with melanoma who received anti-CTLA4 antibody (3 mg/kg every 3 weeks) in combination with antimelanoma peptide vaccines. Three patients experienced objective cancer regression, and two patients experienced mixed responses.10 Grade 3/4 autoimmune toxicities were seen in six (43%) of 14 patients. Further trials established that tumor regression could also be seen without added vaccination. A number of grade 3/4 immune-mediated toxicities, unanticipated by preclinical testing in nonhuman primates, were encountered in patients given ipilimumab.11-13 These included dermatitis, enterocolitis, hypophysitis, uveitis, and hepatitis. Mice with their CTLA4 genes knocked out show lethal lymphoproliferation as well as myocarditis and pancreatitis.14 Administration of anti-CTLA4 antibody in mice also enhanced experimental autoimmune myasthenia gravis,15 precipitated and exacerbated autoimmune diabetes16 and experimental autoimmune encephalomyelitis,17 and induced autoimmune gastritis.18 Population-based studies found that specific polymorphisms in the human CTLA4 gene were associated with increased risks of autoimmune diabetes and thyroid disease.19 Therefore, these ipilimumab-associated toxicities were thought to be possible autoimmune manifestations of CTLA4 blockade. To further investigate this hypothesis, we studied the most frequent ipilimumab-associated toxicity, enterocolitis, to determine its clinicopathologic characteristics, contributing factors, response to therapy, and association with tumor regression. A total of 234 patients with metastatic melanoma (MM) or renal cell carcinoma (RCC) have received ipilimumab in the Surgery Branch of the National Cancer Institute. One hundred thirty-seven of these patients had melanoma and received antibody with or without melanoma peptide vaccines. Sixty-one patients with metastatic clear-cell RCC were given ipilimumab without vaccination. Thirty-six additional patients receiving ipilimumab in combination with high-dose interleukin-2 (IL-2) are not included in this report. Enterocolitis was the most frequent significant adverse occurrence, but we also observed dermatitis, hypophysitis, uveitis, hepatitis, nephritis, and one case of autoimmune meningitis. This report presents the clinicopathologic results and outcome analysis on the 41 patients who developed enterocolitis in association with ipilimumab treatment.
Patients One hundred ninety-eight patients were treated with intravenous human immunoglobulin anti-CTLA4 monoclonal antibody ipilimumab, from March 19, 2002, to July 15, 2005. All patients had a histologic diagnosis of stage IV cutaneous melanoma or stage IV clear-cell RCC, and had measurable disease. All patients had a life expectancy  3 months and an Eastern Cooperative Oncology Group performance status  2. A normal CBC, creatinine, hepatic panel, hepatitis, HIV, and autoimmunity screen was required. Patients receiving a peptide vaccine were constrained to be human leukocyte antigen (HLA) -A0201–positive. Patients with RCC were either IL-2 refractory or IL-2 ineligible. Patients with any other major malignancy, a history of autoimmune disease, a requirement for immunosuppressive agents, who were pregnant or nursing, or who had received prior ipilimumab treatment were excluded.
Treatment With Ipilimumab Melanoma patients treated with ipilimumab + vaccines in the first protocol received ipilimumab at a dose level of 3 mg/kg for all doses in cohort I or an initial loading dose of ipilimumab at 3 mg/kg, followed by all subsequent doses at 1 mg/kg in cohort II.
Melanoma patients treated with ipilimumab dose escalation ± vaccines were treated with intrapatient dose escalation until objective clinical response, The treatment regimen for RCC consisted of two cohorts: cohort I received MDX at 3 mg/kg with subsequent doses of 1 mg/kg every 3 weeks and cohort II were treated with 3 mg/kg every 3 weeks for all doses. Diagnosis of Enterocolitis Assessment of enterocolitis varied in the first 10 presenting patients. One additional patient presented with colonic perforation and no antecedent symptoms. The remaining 30 patients were admitted for work-up on onset of diarrhea, stopped oral intake, and were given intravenous hydration. Stool was sent for standard microbiological and parasite examinations in order to rule out an infectious etiology. Colonoscopy or flexible sigmoidoscopy with biopsies was performed in 40 of the 41 patients. Many patients also underwent esophagogastroduodenoscopy with biopsies. Patients were considered to have enterocolitis if they had either biopsy findings showing enterocolitis or the clinical scenario of sudden onset diarrhea, no alternate etiology identified, and a response to steroid therapy.
Treatment Regimen for Enterocolitis
Evaluation of Clinical Response to Ipilimumab
Response was evaluated using Response Evaluation Criteria in Solid Tumors. A partial response (PR) was defined as a
Statistical Analysis
Patient Characteristics A total of 137 patients with melanoma received ipilimumab; 56 patients were treated with ipilimumab + vaccines and 81 patients with escalating doses of ipilimumab with HLA-A0201 patients randomized to receive or not receive vaccines. Sixty-one patients with RCC were treated with ipilimumab alone (Table 1).
Enterocolitis Forty-one patients were diagnosed with enterocolitis for an overall incidence of 21%. Incidence by protocol was as follows: 14% for ipilimumab + vaccines for melanoma, 20% for ipilimumab dose escalation ± vaccines for melanoma, and 28% for RCC. There was no significant difference in incidence of enterocolitis between protocols (all pairwise comparisons between protocols NS, P > .1) nor between those patients who received 3 mg/kg per dose of ipilimumab and those who received doses of 5 mg/kg (Table 1). The difference between all patients with melanoma versus those with RCC was also not statistically significant (P = .128).
Presenting Symptoms
The median number of days of symptoms on admission was found to be 5 (range, 1 to 64 days, data available on 37 patients) with 86% of patients presenting within 7 days of onset. For the 39 patients with available data, the median number of days from the last dose of ipilimumab to the onset of symptoms was 11 (range, 0 to 59 days). All but four patients developed symptoms within 21 days of their last dose (Fig 1). Patients received between one and 10 doses of ipilimumab before the onset of enterocolitis, with no predictable pattern (Fig 2).
Endoscopic Findings Of the 40 patients who underwent flexible sigmoidoscopy or colonoscopy, reported gross findings were available for 36. Twenty-three had erythema or ulceration. Thirty-six of 40 patients had histologically proven colitis, including all 23 patients with gross abnormalities. One additional patient (the only one without diarrhea) was diagnosed with colitis retrospectively from a colectomy specimen after perforating. Eighteen patients also underwent esophagogastroscopy, and reports of gross findings were available for 16. Ten patients had grossly positive endoscopies. Fourteen patients had histologically proven gastritis or duodenitis on pathological review, including two with histologically negative colonic biopsies. In total, 39/41 patients had histologically proven enterocolitis. Two patients, including one patient with perforation, did not have histologic findings of enterocolitis, but were diagnosed by their clinical course.
Histolopathologic Features of Enterocolitis
Treatment Thirty-four of the 41 patients were treated with corticosteroids. The median time between onset of symptoms and initiation of steroid therapy was available for 31 patients and was found to be 8 days (range, one to 66 days). One patient who developed symptoms 66 days before steroid therapy first had spontaneous improvement, was re-treated with ipilimumab, and then relapsed. Twelve patients treated with steroids had refractory enterocolitis as defined by a failure to respond to steroid therapy within 7 days (five patients) or an initial response to steroids followed by a relapse requiring reinstitution of high-dose steroids (seven patients). Seven patients were not treated with steroids because enterocolitis developed before establishing a consistent steroid based treatment regimen (one patient) the initial presentation was perforation (one patient), mild symptoms rapidly resolved spontaneously (four patients), or infliximab alone was effective (one patient). Four additional patients with enterocolitis refractory to high-dose steroids (more than 10 to 69 days) received a single dose of infliximab at 5 mg/kg as second-line therapy, and all showed rapid and durable resolution of symptoms (Table 3).
Complications Four patients experienced colonic perforation secondary to enterocolitis, three with RCC, and one with melanoma. Three of these perforations were in patients who were refractory to their initial treatment with high-dose steroids. Perforation occurred after one, four, six, and six doses of ipilimumab, respectively. Two patients died after perforation—one with overwhelming sepsis and one electing for comfort care due to cancer progression. One additional patient with RCC required colectomy for persistent gastrointestinal bleeding secondary to steroid refractory enterocolitis. Thus, the incidence of perforation or colectomy in patients being treated for RCC was 6.6% (four of 61 patients) and 0.7% (one of 137 patients) in patients with melanoma (P = .032). The mortality rate among patients who developed enterocolitis was 5% (two of 41 patients). The mortality rate among all treated patients was 1% (two of 198 patients).
Other Immune Mediated Toxicities
Clinical Response to Ipilimumab
Several clinical studies now confirm that ipilimumab, an antibody to human CTLA4, can cause the durable regression of both MM and RCC, even in patients who had previously not responded to other immunotherapies such as interleukin-2. This antibody also induces immune-mediated toxicities in several normal organs and tissues, including the upper and lower gastrointestinal tract, anterior pituitary, skin, uveal tract, and liver. Of these sites, major toxicity was seen most frequently in the gastrointestinal tract. Overall, 21% of all patients receiving ipilimumab developed stage III/IV clinical enterocolitis or had biopsy confirmation of enterocolitis. It was seen in patients with melanoma as well as RCC, though the latter patients had a higher frequency of severe complications. An acute histological picture with neutrophilic infiltrates as well as a chronic picture with infiltrating lymphocytes, and even granulomata, can be seen and does not help to define the pathophysiology. The clinical hallmark is diarrhea, which responds rapidly to withholding oral feedings, and in most cases, high-dose steroids. The enterocolitis associated with ipilimumab has features similar to both graft-versus-host disease as well as inflammatory bowel disease. Enterocolitis from graft-versus-host disease was significantly associated with the regression of RCC in patients treated with a mini-allotransplant regimen.20 A randomized study has suggested that a contributing factor to enterocolitis in this setting may be intestinal microflora and bacterial antigens,21,22 and this may be an area of future investigation for prophylaxis of enterocolitis after ipilimumab. The enterocolitis after ipilimumab is also similar to inflammatory bowel disease in its clinical picture of acute and chronic inflammatory changes, skip areas, and its response to infliximab. Recent data have implicated polymorphisms in the CARD15/NOD2 gene, as well as antibody responses to bacterial antigens as risk factors in developing inflammatory bowel disease.23-25 These factors and known polymorphisms in the CTLA4 gene are currently being investigated in our patients to determine if they affect the risk of developing enterocolitis after receiving ipilimumab. Another potential mechanism for generating enterocolitis after anti-CTLA4 antibody involves CD25+CD4+ regulatory T cells (Treg). These immunosuppressive regulatory cells constitutively express high levels of CTLA4, unlike other T cells, where CTLA4 is only induced after activation.6 Mice lacking Treg cells (through a germline disruption of the FoxP3 gene) and patients with the IPEX syndrome (immune-dysfunction, polyendocrinopathy, enteropathy, and X-linked inheritance) who have a mutation in FoxP3, both show autoimmune disease.26-28 Furthermore, transfer of CD25+CD4+ T cells into mice with an experimental immune-mediated colitis leads to resolution of colitis.29 Therefore, it has been postulated that antibody to CTLA4 might deplete Treg cells and thus induce autoimmunity. However preliminary data from our patients receiving ipilimumab have not shown a decrease in Treg number or function in peripheral blood after ipilimumab.30 Nevertheless, examination of Treg cells in tumor or other relevant tissues may be needed to fully investigate this issue. A short course of high-dose steroids was a definitive treatment for enterocolitis in the majority of our patients. Reinstituting steroids successfully treated most of those suffering a relapse, but infliximab also seemed to be an effective therapeutic option in four patients with steroid-refractory enterocolitis. Further investigation into the impact of tumor necrosis factor alpha neutralization on the antitumor response is warranted before routinely recommending infliximab in this setting. The mortality in patients who developed autoimmune colitis was 5%. Several patients with protracted colitis and major complications either did not receive prompt treatment or were poorly compliant in taking steroids. Instruction in symptom recognition, timely diagnostic studies and prompt treatment with high-dose steroids with compliance monitoring may reduce the risks of colonic perforation, bleeding, or death. The 2.5% overall risk of perforation or colectomy and the 1% overall risk of death should be weighed against the 14% response rate, often of significant duration, in patients with widespread melanoma or RCC. One of the most intriguing findings was the association between enterocolitis and tumor regression. Enterocolitis could be a surrogate marker of drug efficacy, or there may be a causal relationship. There could be common antigens expressed by tumor and bowel, though they would have to be found on both melanoma and RCC. It is also possible that immune-mediated enterocolitis nonspecifically supports an independent antitumor immune response, either through cytokine production or dendritic cell activation by CD4 cells of autoimmune origin. Alternatively, there could be a genetic predisposition in responding patients for autoimmunity that extends to enterocolitis and tumor rejection mediated by "self-antigens." These hypotheses require further laboratory investigation. Clearly, amelioration of this significant toxicity would facilitate the utilization of ipilimumab, an active immunotherapeutic reagent that has shown promise in treating melanoma and RCC. A better understanding of the significant role of CTLA4 in tumor tolerance should also lead to new approaches and immunological strategies for inducing durable tumor rejection.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C)
Supported in part by the Intramural Research Program of the National Cancer Institute, National Institutes of Health. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Brunet JF, Dosseto M, Denizot F, et al: The inducible cytotoxic T-lymphocyte-associated gene transcript CTLA-1 sequence and gene localization to mouse chromosome 14. Nature 322:268-271, 1986[CrossRef][Medline] 2. Walunas TL, Lenschow DJ, Bakker CY, et al: CTLA-4 can function as a negative regulator of T cell activation. Immunity 1:405-413, 1994[CrossRef][Medline] 3. Krummel MF, Allison JP: CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J Exp Med 182:459-465, 1995 4. Krummel MF, Allison JP: CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells. J Exp Med 183:2533-2540, 1996 5. Chambers CA, Krummel MF, Boitel B, et al: The role of CTLA-4 in the regulation and initiation of T-cell responses. Immunol Rev 153:27-46, 1996[CrossRef][Medline] 6. Read S, Malmstrom V, Powrie F: Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med 192:295-302, 2000 7. Leach DR, Krummel MF, Allison JP: Enhancement of antitumor immunity by CTLA-4 blockade. Science 271:1734-1736, 1996[Abstract] 8. van Elsas A, Hurwitz AA, Allison JP: Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med 190:355-366, 1999 9. Hodi FS, Mihm MC, Soiffer RJ, et al: Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients. Proc Natl Acad Sci U S A 100:4712-4717, 2003 10. Phan GQ, Yang JC, Sherry RM, et al: Cancer regression and autoimmunity induced by CTLA-4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci U S A 100:8372-8377, 2003 11. Keler T, Halk E, Vitale L, et al: Activity and safety of CTLA-4 blockade combined with vaccines in cynomolgus macaques. J Immunol 171:6251-6259, 2003 12. Sanderson K, Scotland R, Lee P, et al: Autoimmunity in a phase I trial of a fully human anti-cytotoxic T-lymphocyte antigen-4 monoclonal antibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma. J Clin Oncol 23:741-750, 2005 13. Attia P, Phan GQ, Maker AV, et al: Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4. J Clin Oncol 23:6043-6053, 2005 14. Tivol EA, Borriello F, Schweitzer AN, et al: Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541-547, 1995[CrossRef][Medline] 15. Wang HB, Shi FD, Li H, et al: Anti-CTLA-4 antibody treatment triggers determinant spreading and enhances murine myasthenia gravis. J Immunol 166:6430-6436, 2001 16. Luhder F, Hoglund P, Allison JP, et al: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulates the unfolding of autoimmune diabetes. J Exp Med 187:427-432, 1998 17. Perrin PJ, Maldonado JH, Davis TA, et al: CTLA-4 blockade enhances clinical disease and cytokine production during experimental allergic encephalomyelitis. J Immunol 157:1333-1336, 1996[Abstract] 18. Takahashi T, Tagami T, Yamazaki S, et al: Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med 192:303-310, 2000 19. Ueda H, Howson JM, Esposito L, et al: Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423:506-511, 2003[CrossRef][Medline] 20. Childs R, Chernoff A, Contentin N, et al: Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N Engl J Med 343:750-758, 2000 21. Beelen DW, Elmaagacli A, Muller KD, et al: Influence of intestinal bacterial decontamination using metronidazole and ciprofloxacin or ciprofloxacin alone on the development of acute graft-versus-host disease after marrow transplantation in patients with hematologic malignancies: Final results and long-term follow-up of an open-label prospective randomized trial. Blood 93:3267-3275, 1999 22. Guthery SL, Heubi JE, Filipovich A: Enteral metronidazole for the prevention of graft versus host disease in pediatric marrow transplant recipients: Results of a pilot study. Bone Marrow Transplant 33:1235-1239, 2004[Medline] 23. Hampe J, Cuthbert A, Croucher PJ, et al: Association between insertion mutation in NOD2 gene and Crohn's disease in German and British populations. Lancet 357:1925-1928, 2001[CrossRef][Medline] 24. Targan SR, Landers CJ, Yang H, et al: Antibodies to CBir1 flagellin define a unique response that is associated independently with complicated Crohn's disease. Gastroenterology 128:2020-2028, 2005[CrossRef][Medline] 25. Arnott ID, Landers CJ, Nimmo EJ, et al: Sero-reactivity to microbial components in Crohn's disease is associated with disease severity and progression, but not NOD2/CARD15 genotype. Am J Gastroenterol 99:2376-2384, 2004[CrossRef][Medline] 26. Brunkow ME, Jeffery EW, Hjerrild KA, et al: Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet 27:68-73, 2001[Medline] 27. Bennett CL, Ochs HD: IPEX is a unique X-linked syndrome characterized by immune dysfunction, polyendocrinopathy, enteropathy, and a variety of autoimmune phenomena. Curr Opin Pediatr 13:533-538, 2001[CrossRef][Medline] 28. Bennett CL, Christie J, Ramsdell F, et al: The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27:20-21, 2001[CrossRef][Medline] 29. Mottet C, Uhlig HH, Powrie F: Cutting edge: Cure of colitis by CD4+CD25+ regulatory T cells. J Immunol 170:3939-3943, 2003 30. Maker AV, Attia P, Rosenberg SA: Analysis of the cellular mechanism of antitumor responses and autoimmunity in patients treated with CTLA-4 blockage. J Immunol 175:7746-7754, 2005 Submitted October 20, 2005; accepted January 13, 2006. Related Editorial
Related Correspondence
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
INTRODUCTION
