1 Royal Marsden Hospital, London and Surrey; 2 St Mungo Institute, Glasgow; 3 Southampton Hospitals NHS Trust, Southampton; 4 St James University Hospital, Leeds; 5 Weston Park Hospital NHS Trust, Sheffield; 6 Birmingham Heartlands Hospital, London; 7 Aberdeen Royal Infirmary, Aberdeen; 8 Guy's Hospital, London; 9 Belfast City Hospital, Belfast, UK; 10 National Institute of Haematology and Transfusiology, Sofia, Bulgaria; 11 ISIS Pharmaceuticals, Inc., Carlsbad, CA, USA
* Correspondence to:Professor D. Cunningham, Department of Medicine, Royal Marsden Hospital, Downs Road, Sutton, Surrey SM2 5PT, UK. Tel: +44-20-8661-3156; Fax: +44-20-8643-9414; Email: dcunn{at}icr.ac.uk
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Abstract |
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Patients and methods: Twenty-six patients received ISIS 3521 (2 mg/kg/day) as a continuous infusion over 21 days of each 28-day cycle.
Results: The median age of the patients was 53 years (range 3777). Histological subtypes were low-grade follicular lymphoma (n=22) and B-cell small lymphocytic lymphoma (n=4). Twenty-one (81%) had stage III/IV disease. The median number of previous lines of chemotherapy was two (range one to six). A total of 87 cycles of ISIS 3521 were administered. Twenty-three patients were assessable for response. Three patients achieved a partial response. No complete responses were observed. Ten patients had stable disease. Grade 34 toxicity was as follows: neutropenia (3.8%) and thrombocytopenia (26.9%).
Conclusions: ISIS 3521 has demonstrated anti-tumour activity in patients with relapsed low-grade NHL. There may be a potential role for this agent in combination with conventional chemotherapy for advanced low-grade lymphoma, and further trials are warranted.
Key words: antisense, follicular, non-Hodgkin's lymphoma, treatment
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Introduction |
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Protein kinase C (PKC) is a family of serine threonine protein kinases that are involved in signal transduction pathways regulating proliferation and apoptosis [3]. There are many isoforms of this family, and the isoenzyme PKC-
is found in a number of transformed cell lines [4
, 5
]. It appears to play a pivotal role in tumour growth and progression in certain malignancies [4
]. In vitro experiments have demonstrated that overexpression of PKC-
in the breast cancer cell line MCF-7 leads to increased anchorage-independent growth and proliferation, and a greater degree of tumorigenicity [6
].
Single-stranded antisense DNA forms a covalent bond with a specific sequence of messenger RNA (mRNA) inhibiting translation and rendering the complex susceptible to degradation by the enzyme RNaseH. ISIS 3521 is a 20-base phosphorothioate antisense oligonucleotide targeted to the 3'-untranslated region of the human PKC- mRNA. In vitro experiments in the A549 human non-small-cell lung carcinoma cell line showed that administration of ISIS 3521 caused a significant decrease in PKC-
protein levels [7
9
].
Antitumour activity of ISIS 3521 has been demonstrated in phase I trials. Nemunaitis et al. [10] reported objective complete responses in two patients with NHL receiving ISIS 3521 three times a week as a 2-h infusion, whilst a partial response was observed by Yuen et al. [11
] in a patient with ovarian cancer receiving ISIS 3521 as a continuous infusion over 21 days. The toxicity profile was acceptable with both these schedules of administration. Thrombocytopenia was the dose-limiting toxicity and was more frequent with the continuous infusion, whilst coagulation abnormalities were seen more often with the thrice-weekly regimen. The 21-day infusion regime has been adopted for further studies based on a prolonged rate of tumour exposure to the treatment and fewer hospital visits for the patient. The maximum tolerated dose of ISIS 3521 was 2 mg/kg/day by continuous infusion. This dose was recommended for phase II studies.
This study was therefore performed to evaluate the efficacy and safety of ISIS 3521 given at 2 mg/kg/day in previously treated patients with low-grade NHL.
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Patients and methods |
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Eligibility criteria were: histologically confirmed low-grade, follicular or lymphocytic non-Hodgkin's B-cell lymphoma; documented progressive disease after at least one previous line of chemotherapy; adequate bone marrow reserve (platelets >100 x 109/l, neutrophils >1.0 x 109/l), renal function (serum creatinine <140 µmol/l), hepatic function (serum bilirubin <35 µmol/l, serum aspartase transaminase and alanine transaminase <3x the upper limit of normal), activated partial thromboplastim time (APTT) within normal range and bidimensionally measurable disease; and Eastern Cooperative Oncology Group (ECOG) performance status 02. Patients were excluded if they had an active bleeding disorder, were on therapeutic doses of warfarin or heparin, had a history of other malignancy (apart from curatively treated superficial skin cancers, or carcinoma in situ of the uterine cervix), or if there was central nervous system involvement with lymphoma.
Therapy
ISIS 3521 (5'-GTTCTCGCTGGTGAGTTTCA-3') 2 mg/kg/day was administered as a continuous intravenous infusion over 21 days of each 28-day cycle, followed by a 7-day treatment-free interval. The chemotherapy was infused by a portable pump via a skin-tunnelled central venous catheter. Patients could continue on therapy until there was objective evidence of disease progression or, in the event of complete response, for an additional two cycles.
Toxicity evaluation and dose modifications
Toxicity was evaluated weekly and graded according to the National Cancer Institute Common Toxicity Criteria. For non-haematological toxicity of grade 3 or 4, treatment was suspended until this had resolved to grade <1 and then treatment was reinstituted with 50% dose reduction (1 mg/kg/day). The following dose reductions were made for haematological toxicity: for grade 3 or 4 thrombocytopenia, treatment was suspended until recovery to grade 1 and then treatment was recommenced with 50% dose reduction (1 mg/kg/day); for grade 3 or 4 neutropenia, treatment was stopped until resolution to grade 2 and then restarted with 50% dose reduction (1 mg/kg/day). Any patient not meeting the above recovery criteria within 2 weeks of the last dose of study drug was withdrawn from the study.
Pre-treatment evaluation, assessment during treatment and follow-up
Patients were assessed at baseline with a full medical history and physical examination including ECOG performance status and measurement of any palpable disease, full blood count, coagulation tests, serum biochemistry including electrolytes, hepatic and renal function tests, urinalysis, bone marrow aspirate and trephine, and computed tomography (CT) scans of chest, abdomen and pelvis. Histological samples were reviewed centrally by an independent pathologist.
During the study, medical history, physical evaluation, full blood count, coagulation screen and serum chemistry were obtained every 28 days.
Response assessment
Tumour response was assessed by CT scans (according to World Health Organisation criteria), clinical measurement of palpable tumour masses and bone marrow biopsy where applicable. These tests were repeated after every second cycle of treatment.
A complete response was defined as disappearance of all radiological and clinical evidence of tumour confirmed on two observations at least 4 weeks apart. In addition, a bone marrow biopsy was required to confirm the absence of bone marrow disease and patients must have been free of lymphoma-related symptoms. A partial response was defined as a 50% decrease in the sum of the products of the measurable lesions confirmed on two observations at least 4 weeks apart. No increase in size in any existing lesion or the appearance of any new lesion may have occurred. Bone marrow disease may not have appeared when previously absent. Stable disease was defined as disease less than a partial response, i.e.
50% decrease in the overall sum of the products of the measurable lesions or the absence of progressive disease at 8 weeks. Progressive disease was defined as a
25% increase in the overall sum of the products of measurable lesions or the appearance of a new lesion or the development of bone marrow involvement when previously absent.
Statistics
Patient demographics and baseline characteristics were summarised. Patients who received any study drug were evaluated for toxicities. Adverse events were summarised by COSTART preferred terms and toxicity grades. Patients who completed at least one cycle of treatment were considered evaluable for efficacy. Proportions of patients experiencing complete response, partial response, stable disease and progressive disease were calculated. Median time to progression was estimated using the KaplanMeier method.
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Results |
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Toxicities
All 26 patients were evaluable for toxicities. Common side effects reported were nausea, fever, headache and fatigue. The most commonly reported events are shown in Table 3. Clinically significant haematological toxicities were largely limited to thrombocytopenia. Grade 3 thrombocytopenia requiring dose reduction occurred in four patients (15.4%), and one patient was withdrawn with grade 4 thrombocytopenia. There was one case of grade 3/4 neutropenia encountered. Grade 3/4 lymphopenia was reported in 12 patients. No significant coagulation disturbance was seen. Hepatic toxicity was mild, with no grade 3 or 4 toxicity seen. In three patients, grade 1 renal toxicity (elevation of creatinine) was observed, contributed to by the co-administration of gentamicin in two cases. A further patient discontinued the study drug after developing grade 2 renal toxicity, again occurring in conjunction with the co-administration of gentamicin. During the course of the study, eight venous catheter infections required treatment with intravenous antibiotics.
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Efficacy
Three patients were excluded from response assessment: one patient withdrew voluntarily from the study prior to completion of cycle 1, another patient discontinued the study prior to completion of cycle 1 because of a serious adverse event (renal dysfunction), a further patient had undergone excision of a marker lesion whilst receiving the study drug, thus response assessment was inaccurate in this case. Response data are shown in Table 4. No complete responses were seen. Amongst the patients assessed for response, three (11.5%) had a partial response as their best, 10 (38.5%) achieved stable disease as their best response and 10 (38.5%) had progressive disease. Responses were observed in patients with low-grade follicular lymphoma stage IVB, IIIA and IE. The patients were 38, 59 and 9 months from the time of initial diagnosis, and had all previously received two or more lines of treatment prior to study entry. One patient had relapsed disease following previous treatment with fludarabine, chlorambucil and rituximab; the second responding patient had relapsed disease following previous therapy with CVP (cyclophosphamide, vincristine and prednisolone), fludarabine and CHOP (cyclophosphamide, adriamycin, prednisolone and vincristine); and the third patient had disease that was refractory to both CVP and CHOP chemotherapy. The partial responses occurred at cycle 11, 5 and 2, respectively. The overall median time to progression for all evaluable patients was 2.8 months.
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Discussion |
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Antisense oligonucleotides have been investigated in follicular lymphoma previously. Webb et al. [13] demonstrated that BCL2 antisense caused tumour regression, symptom relief and decreased numbers of circulating lymphoma cells in multiply treated patients with advanced stage low-grade lymphoma. Furthermore, this agent exhibited chemosensitising properties, as several patients treated with chemotherapy after antisense achieved a partial response. Non-antisense-mediated effects may contribute to the clinical activity seen with phosphorothioated antisense oligonucleotides. Preclinical studies have evaluated the immune stimulatory activity of oligonucleotides and have found it to be related to the presence of certain oligonucleotide sequences, in particular the presence of CG dinucleotides [14
]. The oligonucleotide sequence of ISIS 3521 does not contain oligonucleotide sequences with high immunostimulatory properties, although of note the phosphorothioate backbone is known to have some immunostimulatory activity independent of the oligonucleotide sequence [15
]. This study does not allow us to assess the relative contribution of the antisense and non-antisense (immunostimulatory) activity to the clinical outcomes seen.
This study has demonstrated that ISIS 3521 is a feasible treatment for patients with multiply treated low-grade NHL in terms of safety and antitumour activity. Three patients achieved a partial response and a further 10 patients with follicular NHL had stable disease. The median time to progression after treatment with ISIS 3521 was 2.8 months. Furthermore, one of the responders had refractory low-grade NHL having failed to respond to two previous lines of treatment including an anthracycline-containing regimen.
The earliest response was observed after two cycles of treatment in this study, but the other two responders had their first responses at cycles 5 and 11. The median treatment delivery for all study patients was only two cycles, suggesting that a higher response rate may have been observed if patients had received a longer treatment course.
The treatment was generally well tolerated, with no excess or unexpected toxicity. There were a number of treatment-related infections, although there was only one case of febrile neutropenia. Thrombocytopenia was the principle haematological toxicity observed, which appears to be a class effect of oligonucleotide phosphorothiates [1620
], and in this study may be partly accounted for by a large proportion of patients having advanced stage disease and receiving multiple lines of previous chemotherapy. Despite 26.9% of patients experiencing grade 34 thrombocytopenia, there was only one case of severe haemorrhage reported and this occurred in a patient receiving concurrent warfarin medication for a Hickman line thrombosis.
Whilst ISIS 3521 has shown promising antitumour activity in this study, it may be possible to further improve these data by combining this treatment with conventional chemotherapy. This has already been evaluated in a number of other malignancies; a phase I study has recently reported encouraging antitumour activity of ISIS 3521 in combination with 5-fluorouracil and leucovorin in patients with refractory advanced cancer, predominantly of colorectal origin [21]. ISIS 3521 administered with carboplatin and paclitaxel in patients with advanced non-small-cell lung cancer has produced promising efficacy data without incurring any additional toxicity [22
]. Yuen et al. [22
] reported an objective response rate of 42% with this treatment, and only 17% of patients progressed during treatment. These particular data have led to a large randomised phase III study comparing this novel combination with carboplatin and paclitaxel alone in this cohort of patients.
The results achieved in this study with ISIS 3521 are comparable to those reported with antisense oligonucleotides targeting BCL2 in patients with multiply pre-treated follicular lymphoma [12, 13
, 23
]. ISIS 3521 required prolonged central venous access, and the earliest time to response was 2 months. Therefore, we would not recommend its use as monotherapy for these patients. Preclinical data have indicated that ISIS 3521 administered in combination with conventional chemotherapy agents produces a greater antitumour effect than observed with the individual agents alone [24
]. ISIS 3521 may cause tumour cells to be more susceptible to apoptopic injury secondary to cytotoxic agents, and there may be a potential role for ISIS 3521 in combination with conventional chemotherapy for follicular lymphoma. Further trials are warranted.
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Acknowledgements |
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Received for publication January 30, 2004. Revision received May 12, 2004. Accepted for publication May 13, 2004.
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References |
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